Adenosine Triphosphate Response Research Articles

P2Y receptor-induced EDHF vasodilatation is of primary importance for the regulation of perfusion pressure in the peripheral circulation of the rat.

Extracellular nucleotides have been shown to induce vasodilatation of conductance arteries by release of the endothelium-derived hyperpolarizing factor (EDHF). As small resistance arteries are of greater importance for blood pressure regulation, a whole rat mesenteric arterial bed preparation was used in the present study when evaluating the physiological relevance for EDHF in mediating nucleotide dilatation. Dilatory responses were examined after pre-contraction with noradrenaline in the presence of 10 mM indomethacin. Adenosine-5'-O-thiodiphosphate (ADPbetaS), adenosine triphosphate (ATP) and uridine triphosphate (UTP) induced vasodilatation (pEC50=6.5-7 and E(max)=40-70%), while uridine diphosphate (UDP) was ineffective. Endothelium-derived hyperpolarizing factor was studied in the presence of 0.5 mM Nvarpi-nitro-L-arginine (L-NOARG). ADPbetaS and UTP induced strong and potent EDHF-dilatations, while ATP only had a weak effect (E(max)=25%). After P2X1 receptor desensitization with 10 microM alphabeta-methylene-adenosine triphosphate, the ATP response was significantly increased (E(max)=65%). Putatively, this could be because of simultaneous activation of both endothelial P2Y receptors and P2X1 receptors on smooth muscle cells, which resulted in the release of EDHF and subsequent hyperpolarization, and depolarization, respectively. Nitric oxide (NO) was studied in the presence of 60 mM K+. ADPbetaS, ATP and UTP induced weak NO dilatations, suggesting a minor role for NO as compared with EDHF. In conclusion, extracellular nucleotides stimulate dilatation by activating P2Y(1) and P2Y(2)/P2Y(4) receptors, but not P2Y(6) receptors. The dilatory responses are mediated primarily by EDHF in the peripheral vascular bed.

Ca(2+) signalling by recombinant human CXCR2 chemokine receptors is potentiated by P2Y nucleotide receptors in HEK cells.

1. Human embryonic kidney (HEK)-293 cells expressing recombinant G alpha(i)-coupled, human CXC chemokine receptor 2 (CXCR2) were used to study the elevation of the intracellular [Ca(2+)] ([Ca(2+)](i)) in response to interleukin-8 (IL-8) following pre-stimulation of endogenously expressed P2Y1 or P2Y2 nucleotide receptors. 2. Pre-stimulation of cells with adenosine 5'-triphosphate (ATP) revealed a substantial Ca(2+) signalling component mediated by IL-8 (E(max)=83 +/- 8% of maximal ATP response, pEC(50) of IL-8 response=9.7 +/- 0.1). 3. 1 microM 2-methylthioadenosine 5'-diphosphate (2MeSADP; P2Y1 selective) and 100 microM uridine 5'-triphosphate (UTP; P2Y2 selective) stimulated equivalent maximal increases in [Ca(2+)](i) elevation. However, UTP caused a sustained elevation, whilst following 2MeSADP [Ca(2+)](i) rapidly returned to basal levels. 4. Both UTP and 2MeSADP increased the potency and magnitude of IL-8-mediated [Ca(2+)](i) elevation but the effects of UTP (E(max) of IL-8 response increased to 50 +/- 1% of the maximal response to ATP, pEC(50) increased to 9.8 +/- 0.1) were greater than those of 2MeSADP (E(max) increased to 36 +/- 2%, pEC(50) increased to 8.7 +/- 0.2). 5. 5. The potentiation of IL-8-mediated Ca(2+) signalling by UTP was not dependent upon the time of IL-8 addition following UTP but was dependent on the continued presence of UTP. Potentiated IL-8 Ca(2+) signalling was apparent in the absence of extracellular Ca(2+), demonstrating the release of Ca(2+) from intracellular stores. 6. Activation of P2Y1 and P2Y2 receptors also revealed Ca(2+) signalling by an endogenously expressed, G alpha(s)-coupled beta-adrenoceptor. 7. In conclusion, pre-stimulation of P2Y nucleotide receptors, particularly P2Y2, facilitates Ca(2+) signalling by either recombinant CXCR2 or endogenous beta-adrenoceptors.

AUGMENTED STRETCH ACTIVATED ADENOSINE TRIPHOSPHATE RELEASE FROM BLADDER UROEPITHELIAL CELLS IN PATIENTS WITH INTERSTITIAL CYSTITIS

Extracellular adenosine triphosphate (ATP) has been shown to mediate inflammation and nociception and, therefore, it may have a role in symptoms associated with interstitial cystitis. We theorized that the bladder uroepithelium releases ATP in response to stretch and, furthermore, this process is augmented in interstitial cystitis. We quantitated ATP using the luciferin-luciferase assay. Urinary ATP levels were compared in 35 patients with interstitial cystitis and in 33 normal controls after pH correction. Cultured interstitial cystitis and normal urothelial cells from the bladder biopsies of 5 patients each were stretched with the Flexcell 2000 machine (Flexcell International Corp., McKeesport, Pennsylvania) and supernatant ATP concentrations were measured. Mean urinary ATP plus or minus standard error of mean was significantly higher in patients with interstitial cystitis than in controls (L value 985 +/- 161 versus 377 +/- 27, p = 0.0007). Supernatant ATP released by stretched interstitial cystitis cells was stretch intensity dependent when comparing 0%, 10% and 20% elongation, and was also significantly higher in stretched interstitial cystitis than in stretched normal cells. Adenosine triphosphate was significantly elevated in the urine of individuals with interstitial cystitis and the stretch activated release of ATP was augmented in interstitial cystitis urothelium. Increased extracellular ATP may have a role in mechanosensory transduction and to our knowledge it represents a novel hypothesis.

Stretch-activated release of adenosine triphosphate by bladder uroepithelia is augmented in interstitial cystitis

Extracellular adenosine triphosphate (ATP) has been shown to mediate inflammation and nociception, and it may play a role in the etiology of interstitial cystitis (IC). We theorize that the bladder uroepithelia release ATP in response to stretch and furthermore, this process is augmented in IC.

Expression of P2 receptors in bone and cultured bone cells

Extracellular nucleotides acting through P2 receptors elicit a wide range of responses in many cell types. There is increasing evidence that adenosine triphosphate (ATP) may function as an important local messenger in bone and cartilage. In this study, we used immunocytochemistry, employing novel polyclonal antibodies against P2X 1–7 receptors, and in situ hybridization, using oligonucleotide probes corresponding to P2X 2,4 and P2Y 2,4 messenger RNAs (mRNAs), to localize P2 receptors on undecalcified bone sections and on cultured osteoblasts and osteoclasts. We provide the first direct evidence that the P2X 2 receptor subtype is expressed on osteoclasts, osteoblasts, and chondrocytes. We also obtained evidence for the expression of P2X 5 and P2Y 2 receptors on osteoblasts and chondrocytes, and for P2X 4 and P2X 7 receptors on osteoclasts. Our results confirm earlier reports of P2Y 2 and P2X 4 expression in human osteoclastoma and rabbit osteoclasts, respectively, and are consistent with ATP responses observed on bone cells using electrophysiological techniques. Our novel finding that P2X 2 is expressed by osteoclasts is of particular interest. P2X 2 is the only P2 receptor subtype that requires extracellular acidification to show its full sensitivity to ATP, and our recent functional studies have shown that the stimulatory action of ATP on resorption pit formation by mature osteoclasts is amplified greatly at low pH. These findings point to fundamental new mechanisms for the local modulation of bone resorption.

Vasoconstriction of intrapulmonary arteries to P2-receptor nucleotides in normal and pulmonary hypertensive newborn piglets.

1. The vasoconstrictor responses of isolated intrapulmonary arteries (IPA) to P2-receptor agonists was investigated during adaptation to extrauterine life in the normal piglet and the effect of pulmonary hypertension was studied following exposure of newborn animals to chronic hypobaric hypoxia (51 kPa) for 3 days. 2. At resting tone, alpha,beta-methyleneATP (alpha,beta-meATP) (P2X-receptor agonist) contracted intrapulmonary arteries from adult, but not immature pigs, and repeated application desensitized the response. 3. Adenosine 5'-triphosphate (ATP) induced endothelium-independent relaxation at low concentrations at all ages, a variable contractile response to high concentrations developed by 3 days, becoming larger and consistent by 14 days of age. 4. Uridine 5'-triphosphate (UTP) evoked a contractile response in normal intrapulmonary arteries from foetal to adult life, the magnitude of the response increasing with age. Endothelial removal and pre-incubation with Nomega-nitro-L-arginine methyl ester (L-NAME) (100 microM) increased the contractile response of adult vessels. 5. Pre-incubation with alpha,beta-meATP (100 microM), increased the contractile response to UTP in both newborn and adult vessels. ATP-induced relaxations were reduced in newborn vessels but there was no effect on the responses of adult vessels. 6. Responses to UTP, ATP and alpha,beta-meATP of intrapulmonary arteries from newborn piglets exposed to chronic hypobaric hypoxia for 3 days were normal. 7. In summary, UTP elicited marked vasoconstriction of porcine IPA at all ages. UTP and ATP responses were consistent with activation of the P2Y4-receptor recently identified in vascular smooth muscle by others. alpha, beta-meATP induced a small vasoconstriction in the adult probably via the P2X1-receptor. Responses remained normal in neonatal pulmonary hypertension.

On the mechanism of the enhancement of delayed rectifier K+ current by extracellular ATP in guinea-pig ventricular myocytes.

The effects of extracellular adenosine 5'-triphosphate (ATP) on the delayed rectifier K+ current (IK) were studied in guinea-pig ventricular myocytes using the whole-cell voltage-clamp technique. ATP increased IK concentration dependently with a concentration eliciting a half-maximal response of 1.86 microM and a maximal increase of about 1.8-fold. The enhancement of IK developed slowly, the effect reaching a maximum in about 1.6 min after application of ATP. The rank order of agonist potency in enhancing IK was 2-methylthio-ATP>/= ATP>>alpha,beta-methylene-ATP. The ATP response was attenuated in guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS)- loaded cells, but was not affected by pertussis toxin (PTX)-pre-treatment, indicating that a PTX-insensitive G protein is involved in the response. These features are consistent with operation of P2Y-type purinoceptors. ATP produced a further increase in IK stimulated maximally either by isoprenaline (1 microM) through protein kinase A (PKA) or by 12-O-tetradecanoylphorbol 13-acetate (TPA, 100 nM) through protein kinase C (PKC), while 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7, 10 microM) did not affect the ATP response, suggesting that PKA and PKC do not mediate the response. ATP irreversibly enhanced IK in cells loaded with adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS, 5 mM) or okadaic acid (10 microM), a phosphatase inhibitor, suggesting that a phosphorylation step is present after the receptor stimulation. Genistein, an inhibitor of tyrosine phosphorylation, suppressed the ATP response significantly, while daidzein, an inactive analogue of genistein, had little effect on it, although both genistein or daidzein alone decreased IK. It is hypothesized that tyrosine phosphorylation plays a role in the signalling pathway involved in the enhancement of cardiac IK by P2Y-purinergic stimulation.

ATP reception and chemosensory adaptation in Tetrahymena thermophila.

Micromolar concentrations of adenosine triphosphate (ATP) and its non-hydrolyzable analog &bgr;- &ggr; -methylene ATP are both effective depolarizing chemorepellents in Tetrahymena thermophila. Chemorepellent behavior consists of repeated bouts of backward swimming (avoidance reactions) that can easily be quantified to provide a convenient bioassay for purinergic reception studies. Chemosensory adaptation occurs following prolonged exposure (10 min) to the repellents, and cells regain normal swimming behavior. Adaptation is specific since cells that are behaviorally adapted to either ATP or &bgr;- &ggr; -methylene ATP still retain full responsiveness to the chemorepellents GTP and lysozyme. However, cross adaptation occurs between ATP and &bgr;- &ggr; -methylene ATP, suggesting that they involve the same receptor. Behavioral sensitivity to both ATP and &bgr;- &ggr; -methylene ATP is increased by the addition of Na+, but addition of either Ca2+ or Mg2+ dramatically decreases the response to ATP. These ionic effects are correlated with in vivo ATP hydrolysis, suggesting that divalent ions decrease purinergic sensitivity by activating a Ca2+- or Mg2+-dependent ecto-ATPase to hydrolyze the ATP signal. In vivo [32P]ATP binding studies and Scatchard analysis suggest that the behavioral adaptation is due to a decrease in the number of surface binding sites, as represented by decreased Bmax values. All these changes are reversible (de-adaptation) after 12 min in a repellent-free buffer. Electrophysiological analysis showed that both &bgr;- &ggr; -methylene ATP (10 micromol l-1) and ATP (500 micromol l-1) elicited sustained, reversible depolarizations while GTP (10 micromol l-1) produced a transient depolarization, suggesting that the chemosensory response pathways for ATP and GTP reception may differ. There may be separate ATP and GTP receptors since ATP and GTP responses do not cross-adapt and 'cold' (unlabeled) GTP is not a good inhibitor of [32P]ATP binding. These results suggests that T. thermophila possess high-affinity surface receptors for ATP that are down-regulated during chemosensory adaptation. These ATP receptors may act as chemorepellent receptors to enable T. thermophila to recognize recently lysed cells and avoid a possibly deleterious situation. This is the simplest eukaryotic organism to show an electrophysiological response to external ATP.

Differences in responses to norepinephrine and adenosine triphosphate in isolated, perfused mesenteric vascular beds between normotensive and spontaneously hypertensive rats.

The responses to norepinephrine and adenosine 5' triphosphate (ATP) of isolated, perfused mesenteric vascular beds were compared between spontaneously hypertensive rats (SHRs) and Wistar-Kyoto (WKY) rats. Norepinephrine (0.01-100 nmol) dose-dependently increased perfusion pressure in the intact bed and the arteries, but not in the veins. The maximal responses in SHRs were larger than those in WKY rats. ATP (0.1-3,000 nmol) increased perfusion pressure in all preparations. The responses of the intact bed and the veins were larger in SHRs, whereas there was no strain difference in the arteries. Indomethacin (5 x 10(-6) M) enlarged the norepinephrine responses of both strains only in the intact beds and did not affect the ATP responses, except the veins in SHRs, where it was reduced. N(G)-nitro-L-arginine methyl ester (5 x 10(-6) M), in combination with indomethacin, potentiated the responses, except the arterial response to high doses of norepinephrine in SHRs, which was not affected. Endothelium denudation in the arteries produced similar changes to those after the combined treatment. UK14,304-induced and ADPbetaS-induced decreases in perfusion pressure at increased tone were similar between the strains. Thus neither the vasodilation induced by the stimulation of alpha2-adrenoceptors nor of P2y receptors seems to affect the response to norepinephrine or to ATP, respectively. These results demonstrate that the intact mesenteric vascular bed of SHRs shows potentiated responses not only to norepinephrine, but also to ATP, as compared with WKY rats, and that the critical regions for determining the strain differences for norepinephrine are overall arteries, and that for ATP are the vessels downstream from arterioles. In the intact beds, neither regulation by endogenous prostanoids nor that by endothelium-derived relaxing factors (EDRFs) is implicated in the strain difference. However, these two types of regulation differ markedly between different kinds of vessels.

Purine Nucleotides Induce Regulated Secretion of von Willebrand Factor: Involvement of Cytosolic Ca2+ and Cyclic Adenosine Monophosphate–Dependent Signaling in Endothelial Exocytosis

von Willebrand factor (vWF) is stored and released from endothelial secretory granules called Weibel-Palade (WP) bodies. Acute release can be induced by thrombin, histamine, and other mediators of thrombosis or inflammation. Their effect is thought to be mediated by an increase in intracellular free calcium ([Ca2+]i). Purine nucleotides such as adenosine triphosphate (ATP) and adenosine diphosphate (ADP) are released from platelet dense granules and from ischemic tissues and are important regulators of platelet function and vascular tone. In the present study, we investigated whether they could also induce exocytosis from cultured endothelial cells. ATP (1 to 100 micromol/L) induced a dose-related increase in vWF release, with a 2.3-fold maximal increase after 30 minutes. Similar responses were observed with ADP. ATP induced calcium mobilization from intracellular stores, an effect mimicked by 2-methylthio-ATP, a selective agonist for P2y receptors. However, 2-methylthio-ATP-induced vWF release was only 43% of the ATP response. ATP-induced vWF release was also associated with a twofold increase in cellular cyclic adenosine monophosphate (cAMP) content, and was potentiated by 3-isobutyl-1-methylxanthine ([IBMX] added to increase cAMP levels by blocking cellular phosphodiesterases) and 8-bromo-cAMP and inhibited by more than 50% by Rp-8-CPT-cAMPS, a competitive protein kinase A inhibitor. Adenosine but not 2-methylthio-ATP mimicked the ATP-induced increase in cAMP. ATP-induced vWF release was partly inhibited by adenosine deaminase, which degrades adenosine generated from ATP in the incubation medium. Adenosine (1 to 100 micromol/L) failed to induce vWF release, but potentiated the secretory response to 2-methylthio-ATP and thrombin without modifying the calcium response to these agents. Our results suggest that ATP/ADP can induce vWF release from endothelial cells via dual activation of P2y and adenosine A2 receptors. ATP/ADP-induced exocytosis could be involved in the regulation of thrombus formation and ischemia-reperfusion injuries. Further, we provide evidence that a receptor-mediated increase in cellular cAMP can potentiate the secretory response to calcium-mobilizing agents.

Purine Nucleotides Induce Regulated Secretion of von Willebrand Factor: Involvement of Cytosolic Ca2+ and Cyclic Adenosine Monophosphate–Dependent Signaling in Endothelial Exocytosis

Abstractvon Willebrand factor (vWF) is stored and released from endothelial secretory granules called Weibel-Palade (WP) bodies. Acute release can be induced by thrombin, histamine, and other mediators of thrombosis or inflammation. Their effect is thought to be mediated by an increase in intracellular free calcium ([Ca2+]i). Purine nucleotides such as adenosine triphosphate (ATP) and adenosine diphosphate (ADP) are released from platelet dense granules and from ischemic tissues and are important regulators of platelet function and vascular tone. In the present study, we investigated whether they could also induce exocytosis from cultured endothelial cells. ATP (1 to 100 μmol/L) induced a dose-related increase in vWF release, with a 2.3-fold maximal increase after 30 minutes. Similar responses were observed with ADP. ATP induced calcium mobilization from intracellular stores, an effect mimicked by 2-methylthio-ATP, a selective agonist for P2y receptors. However, 2-methylthio-ATP–induced vWF release was only 43% of the ATP response. ATP-induced vWF release was also associated with a twofold increase in cellular cyclic adenosine monophosphate (cAMP) content, and was potentiated by 3-isobutyl-1-methylxanthine ([IBMX] added to increase cAMP levels by blocking cellular phosphodiesterases) and 8-bromo-cAMP and inhibited by more than 50% by Rp-8-CPT-cAMPS, a competitive protein kinase A inhibitor. Adenosine but not 2-methylthio-ATP mimicked the ATP-induced increase in cAMP. ATP-induced vWF release was partly inhibited by adenosine deaminase, which degrades adenosine generated from ATP in the incubation medium. Adenosine (1 to 100 μmol/L) failed to induce vWF release, but potentiated the secretory response to 2-methylthio-ATP and thrombin without modifying the calcium response to these agents. Our results suggest that ATP/ADP can induce vWF release from endothelial cells via dual activation of P2y and adenosine A2 receptors. ATP/ADP-induced exocytosis could be involved in the regulation of thrombus formation and ischemia-reperfusion injuries. Further, we provide evidence that a receptor-mediated increase in cellular cAMP can potentiate the secretory response to calcium-mobilizing agents.

Administration of Pegylated Recombinant Human Megakaryocyte Growth and Development Factor to Humans Stimulates the Production of Functional Platelets That Show No Evidence of In Vivo Activation

This report describes the effect of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) on platelet production and platelet function in humans. Subjects with advanced solid tumors received PEG-rHuMGDF daily for up to 10 days. There was no increase in circulating platelet count at doses of 0.03 or 0.1 microgram/kg/d by day 12 of study. At doses of 0.3 and 1.0 microgram/kg/d there was a threefold median increase (maximum 10-fold) in platelet count by day 16. The platelets produced in vivo in response to PEG-rHuMGDF showed unchanged aggregation and adenosine triphosphate (ATP)-release responses in in vitro assays. Tests included aggregation and release of ATP in response to adenosine diphosphate (ADP) (10, 5, 2.5, and 1.25 mumol/L), collagen (2 micrograms/mL), thrombin-receptor agonist peptide (TRAP, 10 mumol/L) and ristocetin (1.5 mg/mL). Administration of aspirin to an individual with platelet count of 1,771 x 10(3)/L resulted in the typical aspirin-induced ablation of the normal aggregation and ATP-release response to stimulation with arachidonic acid (0.5 mg/mL), collagen, and ADP (2.5 and 1.25 mumol/L). There was no change in the expression of the platelet-surface activation marker CD62P (P-selectin) nor induction of the fibrinogen binding site on glycoprotein IIb/IIIa as reported by the monoclonal antibody, D3GP3. An elevation of reticulated platelets was evident after 3 days of treatment with PEG-rHuMGDF and preceded the increase in circulating platelet count by 5 to 8 days; this reflected the production of new platelets in response to PEG-rHuMGDF. At later time points, the mean platelet volume (MPV) decreased in a manner inversely proportional to the platelet count. Levels of plasma glycocalicin, a measure of platelet turnover, rose 3 days after the initial increase in the peripheral platelet count. The level of plasma glycocalicin was proportional to the total platelet mass, suggesting that platelets generated in response to PEG-rHuMGDF were not more actively destroyed. Thus, the administration of PEG-rHuMGDF, to humans, increased the circulating platelet count and resulted in fully functional platelets, which showed no detectable increase in reactivity nor alteration in activation status.

The biphasic response of rat vesical smooth muscle to ATP

1. Adenosine-5'-triphosphate (ATP) is known to exert a variety of biological effects via the activation of either ionotropic P2x- or G-protein coupled P2Y-purinoceptor subtypes. In this study the effects induced by ATP and ATP analogues on rat bladder strips were characterized at resting tone and in carbachol-prestimulated tissues. 2. ATP exerted a clear concentration-dependent biphasic response, which was maximal at 1 mM concentration and was characterized by an immediate and transient contraction, followed by a slower sustained relaxation. The receptor mediating contraction was susceptible to desensitization by ATP and by the ATP analogue, alpha,beta-methyleneATP (alpha,beta-meATP) showing the typical features of the P2x-purinoceptor; conversely, ATP-evoked relaxation did not undergo tachyphylaxis following either ATP or alpha,beta-meATP. 3. The slower and sustained relaxant phase seemed to be due to activation of P2Y-purinoceptors, based on responses obtained with the P2Y agonist, 2-methyl-thioATP (2-meSATP) and, more importantly, based on the clear involvement of the G-proteins. In fact, the G-protein activator, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) significantly potentiated and the G-protein blocking agent, guanosine 5'-O-(2-thio-diphosphate) (GDP beta S) completely abolished the ATP-induced relaxation. No effects were exerted by these two G-protein modulators on the ATP-induced contraction. 4. The relaxant component of the ATP response of bladder tissue was not significantly influenced by nitro-benzyl-thioinosine (NBTI) or by 8-phenyltheophylline (8-PT), suggesting that the contribution of the ATP metabolite adenosine to this response was negligible. Moreover, relaxation evoked by ATP and by the adenosine analogue, 5'-N-ethylcarboxamidoadenosine (NECA) was additive.5. Suramin was unable to modify either the relaxant or the contractile responses of bladder strips to ATP. However, when tested on the concentration-response curve to the slowly hydrolysable P2x-agonist alpha,beta-meATP, a rightward shift was detected, suggesting that ATP contractile responses are mediated by suramine-sensitive P2x-purinoceptors.6. Uridine-5'-triphosphate (UTP) only induced a rapid and concentration-dependent contraction of the rat bladder preparation, which was not desensitized by pre-exposure to alpha,beta-meATP, suggesting that UTP responses were not mediated by the 'classical' P2X-purinoceptor.7. It is therefore concluded that both P2x- and P2y-purinoceptors, which mediate ATP-induced contraction and relaxation, respectively, are present in rat bladder. Moreover, removal of epithelium did not affect ATP-elicited contraction, whereas ATP-induced relaxation was significantly augmented. These data suggest that P2x- and P2Y- purinoceptors are localized in smooth muscle cells and that the relaxant response is probably modulated by excitatory factor(s) released by epithelial cells.

ATP activates cationic and anionic conductances in Schwann cells cultured from dorsal root ganglia of the mouse.

The whole-cell configuration of the patch-clamp technique was used to study membrane responses of mouse Schwann cells in organotypique culture to external application of adenosine 5'-triphosphate (ATP). ATP induced an inward current (IATP) which caused a membrane depolarization. IATP was dose dependent with a Kd of 8.4 mM. ATP analogues had the following relative agonist potency: ATP > ADP approximately alpha-beta methylene ATP. Neither AMP nor adenosine were effective. IATP was reversibly reduced by suramin, a P2 purinoceptor antagonist. Alteration of ionic gradients showed that ATP activated simultaneously cationic (potassium and calcium) and anionic (chloride) conductances. Values of the reversal potentials to ATP suggested the following permeability sequence through the anion channel: SCN- > I- > Br- approximately Cl- > aspartate > isethionate. IATP did not appear to be dependent on intracellular calcium, as inclusion or omission of calcium chelator (BAPTA, 10 mM) in the pipette solution had no effect on IATP. The observed ATP response resembles a P2-mediated response in its kinetics, its relative agonist potency and its blockade by suramin, but differs from known ones by its requirement for high concentrations of ATP and the activation of a cationic as well as an anionic conductance.

Involvement of ETA receptors in the facilitation by endothelin-1 of non-adrenergic non-cholinergic transmission in the rat urinary bladder.

1. Endothelin-1 (ET-1; 3-10 nM) raised the tone of rat bladders bathed in buffer containing atropine (1 microM) plus guanethidine (3.4 microM). In addition, ET-1 potentiated, in a concentration-dependent fashion (1-10 nM), the contractions evoked by both transmural nerve stimulation and applications of exogenous adenosine 5'-triphosphate (ATP). 2. The threshold concentration of ET-1 required to facilitate non-adrenergic non-cholinergic (NANC) transmission and potentiate ATP-induced contractions, was about 10 fold lower than that required to increase the bladder tone (3 nM). 3. The ET-1-induced increase in basal tension reached its maximal effect within 60-90 s. In contrast, the 7.8 microM ATP-induced contractions increased by 50% within the first minute following incubation with 10 nM ET-1 but required about 5 min to develop the maximal effect. 4. The ET-1-induced potentiation of NANC or ATP responses was long-lasting and persisted in spite of extensive washing. The recovery of the bladder excitability depended on the concentration of ET-1. Following the application of 3 nM ET-1, recovery required 30 min; applications of 10 nM ET-1 required at least 60 min for full recovery. 5. The ET-1-induced potentiation of responses was selective for ATP and related structural analogues. ET-1 did not modify the contractions induced by acetylcholine, 5-hydroxytryptamine, prostaglandin F2 alpha or bradykinin. 6. The potency of ET-2 was similar to that of ET-1. ET-3 and ET-C-terminal hexapeptide were inactive up to 100 M. Sarafotoxin S6b was 2 to 3 fold less potent than ET-1 whereas sarafotoxin S6c (100 nM) was inactive. AGETB-9 and AGETB-89, two ETB receptor agonists, were also inactive (up to 100 nM). 7. Removal of one or both disulphide bonds in ET-1 and tryptophan-21 formylation of ET-1, resulted in inactive peptides (up to 100 nM). 8. The ET-1 receptor antagonists, BE-18257B and FR 139317, blocked both the ET-1-induced rise in tone and the potentiation of ATP responses in a concentration-dependent fashion. FR 139317 was at least 30 fold more potent than BE-18257B. Both antagonists blocked at lower concentrations the ET-1 increase in bladder tone as compared to the ATP potentiation. The antagonism was slowly reversible. 9. Results are consistent with the presence of ETA receptors in the rat bladder, which mediate both actions of ET-1. The interaction of ET-1 with purinergic mechanisms is discussed.

Blockade by glibenclamide of the flow-evoked endothelial release of ATP that contributes to vasodilatation in the pulmonary vascular bed of the rat.

1. The effect of step augmentation of flow rate on the level of adenosine -5'-triphosphate (ATP) measured in the Krebs perfusate was investigated, and the effect of glibenclamide on the release of ATP was tested in the rat pulmonary vascular bed. 2. For flow rates between 10.38 +/- 1.18 and 28.88 +/- 2.08 ml min-1 (n = 8) 1 microM suramin, a P2-purinoceptor antagonist, significantly (P < 0.05) increased vascular resistance under conditions of step augmentation of flow rate. This suggests that endogenous ATP released during increases in flow rate dilates pulmonary vessels. 3. In response to a step augmentation in flow rate from 9.13 +/- 0.97 to 18.3 +/- 1.69 ml min-1 (n = 4) ATP levels were up to 23 fold higher (P < 0.05) for 15 s, and gradually dropped to a level of about half the initial rise. Once the ATP levels had stabilized, another step augmentation of flow rate to 27.00 +/- 3.49 ml min-1 was able to evoke a corresponding increase of ATP release. The ability of the vascular bed to respond with increased ATP release after the initial ATP responses had tapered, demonstrates that the drop in ATP levels after the initial rise is not due to depletion of ATP. Furthermore, the maximal ATP response directly precedes the vasodilatation observed following each jump in perfusion pressure produced with each step increase in flow rate. 4. In response to two 3 fold step augmentations of flow rate (8.41-27.29 ml min-1) spaced 30 min apart there were two increases in the level of ATP which were not significantly different from each other.However, perfusion with 1 microM glibenclamide between the first and the second step augmentation of flow rate (8.08-24.67 ml min-1) significantly (P<0.05; n = 6) blocked the increase in ATP release. This suggests that the release of intracellular ATP is mediated by glibenclamide-sensitive K+ channels.5. A concentration of 1 microM glibenclamide perfused for 30 min was without effect on vascular pressure at constant flow. However, under conditions where flow was augmented in a stepwise manner (between 11.50 and 36.45 ml min-1) perfusing with 1 microM glibenclamide increased vascular resistance (P <0.10).6. It is concluded that flow-induced ATP release is mediated by a glibenclamide-sensitive K+ channel,and that the release of ATP from endothelial cells probably functions to vasodilate the pulmonary vascular bed of the rat.

Nucleotides mobilize intracellular calcium stores of renal proximal cells in primary culture: Existence of a suramin-sensitive mechanism

Changes of intracellular calcium concentrations [Ca 2+] i were measured in primary cultured rabbit proximal convoluted tubules (PCT). A dual-excitation, digital-imaging inverted microscope was used to monitor the fura-2 fluorescence. The basal calcium level was 106 ± 11 nM (n = 36) . The stimulatory effects of adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine were studied. ATP and ADP induced transient increases of [Ca 2+] i ( 1059 ± 115% of the resting level ( n = 29), and 659 ± 134% (n = 10), respectively) by releasing calcium from cytoplasmic stores. Adenosine had less effect ( 279 ± 48% of the resting level, n = 3). In the same conditions the ATP antagonist suramin (100 μM) inhibited the action of ATP and ADP to 231 ± 52% (n = 3), and 308 ± 29% (n = 4) of the resting level, respectively, but did not modify that of adenosine ( 281 ± 72%, n = 3). A pretreatment (500 ng/ml for 2 h at 37°C) of the culture with the toxin of Bordetella pertussis completely blocked the ATP response. Our results are evidence for the presence of a functional suramin-sensitive ATP and ADP puriceptor in cultured renal proximal cells. A pertussis-toxin-sensitive G protein is linked to the transduction mechanism. This receptor is distinct from an adenosine puriceptor also found in the proximal monolayer.

Localization of cholinergic and purinergic receptors on outer hair cells isolated from the guinea-pig cochlea.

Acetylcholine (ACh) and adenosine 5'-triphosphate (ATP) are shown to act in opposing fashion on guinea-pig cochlear outer hair cells (OHCS) via receptors localized within different fluid compartments of the organ of Corti. The cholinergic (efferent) receptors localized at the basal (perilymphatic) region of these cells activated a rapidly desensitizing hyperpolarizing K+ current. In contrast, purinergic (ATP) receptors were localized at the apical (endolymphatic) surface of OHCS and activated a depolarizing nonselective cation current which exhibited inward rectification and lacked desensitization. Localization of the receptors was determined by using whole-cell patch-clamp, by recording onset latencies and response amplitudes to pulses of either ACh or ATP pressure-applied at selected sites along the length of isolated OHCS. Under voltage-clamp at -60 mV, the largest ACh-induced (outward) currents were recorded when ACh was directed at the basal region of the cells. Conversely, the maximum (inward) ATP currents were obtained when ATP was directed toward the apical surface of these cells. Onset latencies increased rapidly from a minimum of approximately 10 ms for either ACh or ATP as the drug pipette was moved away from these optimal sites. The ATP response was antagonized by amiloride in a dose-dependent manner with a KD of approximately 400 microM. The localization of P2-type purinoceptors to the endolymphatic surface of OHCS suggests that ATP mediates a humoral modulation of the mechano-electrical transduction process.

The nucleotide receptors on mouse C2C12 myotubes

1. The response of C2C12 mouse myotubes to stimulation with adenosine triphosphate (ATP) and other nucleotides was studied by measuring changes in membrane potential. 2. A transient hyperpolarization followed by a slowly declining depolarization of the cells was observed in the presence of ATP (10 microM-1 mM). 3. The hyperpolarization was not observed in the absence of external calcium, and was abolished in the presence of tetraethylammonium (20 mM) or the bee toxin, apamin (0.1 microM). The depolarization was reduced under low sodium conditions. 4. A biphasic change in membrane potential was also recorded in the presence of adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) and the pyrimidine uridine triphosphate (UTP), while the ATP derivatives and analogues, adenosine diphosphate, adenosine, alpha,beta-methylene ATP and 2-methylthio ATP and the nucleotides, guanosine triphosphate and cytidine triphosphate, did not affect the membrane potential of the myotubes. 5. The hyperpolarization elicited by ATP gamma S or UTP was also blocked by apamin and abolished under Ca(2+)-free conditions. 6. In contrast to ATP and ATP gamma S, the depolarization evoked by UTP was unaffected under low Na+ and less sensitive to the antagonistic action of suramin. 7. The ATP and UTP responses at maximal concentration were not additive after simultaneous application. ATP elicited a depolarization if applied after UTP, while UTP did not change membrane potential following the application of ATP. 8. The concentration-response curves of the effective nucleotides were shifted to the right in the presence of suramin, suggesting competitive antagonism.9. These results can be explained by the presence of 'nucleotide receptors' mediating the ATP/UTPinduced hyperpolarization and depolarization in C2C12 myotubes. Furthermore, an increase in Na+-conductivity can be exclusively activated by ATP.

Neurotransmitter release from synaptotagmin-deficient clonal variants of PC12 cells.

Synaptotagmin (p65) is an abundant synaptic vesicle protein of neurons and contains regions similar to the regulatory domain of protein kinase C. These domains are thought to be involved in calcium-dependent interaction with membrane phospholipids during exocytosis. To assess the functional role of synaptotagmin, synaptotagmin-deficient clonal variants of PC12 cells were isolated. All of the variant cells released catecholamine and adenosine triphosphate in response to elevated intracellular concentrations of calcium, which suggests that synaptotagmin is not essential for secretion of catecholamine and adenosine triphosphate from PC12 cells.