Complementary role of extracellular ATP and adenosine in ischemic preconditioning in the rat heart

Although adenosine is an important mediator of ischemic preconditioning (IPC), its relative contribution to IPC remains unknown. Because adenosine is formed through the hydrolysis of ATP, the present study investigated the role of ATP and adenosine in IPC. Isolated and buffer-perfused rat hearts underwent IPC by three cycles of 5-min ischemia and 5-min reperfusion before 25 min of global ischemia. The rate-pressure product (RPP) 30 min after reperfusion was taken as an endpoint of functional protection. Interstitial fluid (ISF) adenine nucleotides and adenosine were measured by cardiac microdialysis techniques. Inhibition of IPC-induced recovery of RPP was partial by the adenosine receptor antagonist 8-(p-sulfophenyl)theophylline (SPT; 100 microM) or by the structurally distinct P2Y purinoceptor antagonists suramin (300 microM) or reactive blue (RB; 10 microM) but was additive when SPT was given with suramin or RB. The P2X antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid tetrasodium (50 microM) had no effect on functional protection. The improved functional recovery was not significantly affected by an ecto-5'-nucleotidase inhibitor, alpha,beta-methylene adenosine diphosphate (AMP-CP; 100 microM), alone but was inhibited by AMP-CP plus SPT, suramin, or RB. ISF ATP and adenosine increased temporarily by 10-fold during IPC. AMP-CP augmented the increase in ISF ATP associated with the decrease in ISF adenosine. There was a reciprocal correlation between the ISF concentration of ATP and adenosine in preconditioned hearts. In addition, there was a significant correlation between ISF adenosine and ATP and the inhibitory potency of SPT and suramin or RB against functional protection conferred by IPC. These results suggest that extracellular ATP and adenosine play a complementary role in IPC through P2Y purinoceptors and adenosine receptors, respectively.     

Extracellular ATP facilitates flow-induced vasodilatation in rat small mesenteric arteries

ATP can be released from endothelial cells, and this release is increased by intraluminal flow in blood vessels. In the present study, the effect of extracellular ATP (1 microM) on flow-induced vasodilatation was investigated in isolated and pressurized rat small mesenteric arteries. In the absence of extracellular ATP, only 46% of arteries developed dilatation in response to flow, and this response was both transient and unstable. In marked contrast, with ATP present, all vessels developed a prolonged and stable dilatation in response to flow. Even in the vessels that failed to respond to flow in the absence of ATP, dilatation could be stimulated once ATP was present. The ability of ATP to facilitate flow-induced vasodilatation was mimicked by UTP (1 microM), a P2Y agonist, or 3'-O-(4-benzoyl)benzoyl ATP (BzATP; 10 microM), an agonist for P2X1, P2X7, and P2Y11 purinoceptors. The involvement of P2X7 purinoceptors was further supported by the inhibitory effect of KN-62 (1 microM), a P2X7 antagonist, on the action of BzATP. P2X1 and P2X3 purinoceptors were not involved because their receptor agonist alpha,beta-methylene ATP had no effect. The facilitating effect of ATP on flow dilatation was also attenuated by the combined application of reactive blue 2 (100 microM), a P2Y antagonist, and suramin (100 microM), a nonselective P2X and P2Y antagonist. Furthermore, flow-induced dilatation obtained in the presence of ATP was reproducible. In contrast, in the additional presence of the ectonucleotidase inhibitor ARL-67156 (10 microM), although the first dilatation was normal, the responses to the second and later exposures to flow were greatly attenuated. The nonhydrolyzable ATP analogs adenosine-5'-(3-thiotriphosphate)trilithium salt (1 microM) and adenosine 5'-(beta,gamma-imido) triphosphate tetralithium salt hydrate (10 microM) had similar effects to those of ARL-67156. These data suggest that ATP acts through both P2X and P2Y purinoceptors to facilitate flow-induced vasodilatation and that ectonucleotidases prevent this effect by degrading ATP on the endothelial cell surface.     

Pancreatic beta-cells communicate via intermittent release of ATP

The role of external ATP for intercellular communication was studied in glucose-stimulated pancreatic beta-cells isolated from ob/ob mice. Digital image analyses with fura-2 revealed spontaneous transients of cytoplasmic Ca2+ appearing in synchrony in the absence of cell contacts. After removal of slow oscillations with methoxyverapamil, addition of ATP (0.1-100 microM) resulted in prompt firing of a transient, followed by suppression of the generation and synchronization of spontaneously occurring transients. It was possible to trigger transients during the suppressive phase by raising the concentration of ATP. The dual action of ATP was mimicked by ADP or 2-methylthio-ATP but not by AMP or UTP. The number of spontaneous transients and their synchronization were reduced in the presence of the dephosphorylating agent apyrase. Additional evidence that intermittent release of ATP participates in the generation of spontaneous Ca2+ transients was obtained from the suppression observed from use of antagonists of the purinoceptors [suramin (0.3-30 microM), pyridoxalphosphate-6-azophenyl-2,4-disulfonic acid (PPADS; 10-30 microM) and 2-deoxy-N-methyladenosine (MRS 2179; 0.3-30 microM)] or from counteracting beta-cell release of ATP by inhibiting exocytosis with 100 nM epinephrine, 100 nM somatostatin, or lowering the temperature below 30 degrees C. The data indicate that ATP has time-dependent actions (prompt stimulation followed by inhibition) on the generation of Ca2+ transients mediated by P2Y receptors. It is proposed that beta-cells both receive a neural ATP signal with coordinating effects on their Ca2+ oscillations and propagate this message to adjacent cells via intermittent release of ATP combined with gap junction coupling.     

Preconditioning modulates susceptibility to ischemia-induced arrhythmias in the rat heart: the role of alpha-adrenergic stimulation and K(ATP) channels

A new concept of cardioprotection based on the exploitation of endogenous mechanisms is known as ischemic preconditioning (IPC). It has been hypothesized that substances released during brief ischemic stress (e.g. catecholamines) stimulate the receptors and trigger multiple cell signaling cascades. Opening of ATP-sensitive K+ channels [K(ATP)] has been suggested as a possible final step in the mechanisms of protection. In this study, the role of adrenergic activation was tested in Langendorff-perfused rat hearts subjected to test ischemia (TI; 30 min occlusion of LAD coronary artery) by: 1) mimicking IPC (5 min ischemia, 10 min reperfusion) with short-term (5 min) administration of norepinephrine (NE, 1 microM), 15 min prior to TI; 2) blockade with beta- or alpha1-receptor antagonists, propranolol (10 microM) and prazosin (2 microM), respectively, applied 15 min prior to TI during IPC. The role of K(ATP) opening was examined by perfusion with a K(ATP) blocker glibenclamide (10 microM) during IPC. Both IPC and NE-induced PC effectively reduced the incidence of ventricular tachycardia (VT) to 33% and 37%, respectively, vs 100% in the non-PC controls, whereby ventricular fibrillation (VF) was totally abolished by IPC and markedly suppressed by PC with NE (0% and 10%, respectively, vs 70% in the non-PC hearts; P < 0.05). The severity of arrhythmias (arrhythmia score, AS) was also markedly attenuated by both interventions (IPC: AS 1.7 +/- 0.4; NE-PC: AS 1.8 +/- 0.3 vs AS 4.1 +/- 0.2 in the controls; P < 0.05). Protection was not suppressed by propranolol (VT 28%; VF 14%; AS 2.2 +/- 0.6), whereas prazosin reversed the protective effect of PC (VT 83%; VF 67%; AS 4.0 +/- 0.8). Antiarrhythmic protection afforded by NE-PC was abolished by pretreatment of rats with pertussis toxin (25 microg/kg, i.p.) given 48 h prior to the experiments. Glibenclamide did not suppress the IPC-induced protection. In conclusion, the sensitivity of the rat heart to ischemic arrhythmias can be modulated by IPC. Protection is mediated via stimulation of alpha1-adrenergic receptors coupled with Gi-proteins but glibenclamide-sensitive K(ATP) channels do not appear to be involved in the mechanisms of antiarrhythmic protection in this model.     

UTP binding and phosphoinositidase C activation in ampulla from frog semicircular canal

Pyrimidine nucleotide-sensitive phosphoinositidase C activity (PLC), previously identified in frog semicircular canal ampulla, was pharmacologically characterized. Binding of [(3)H]UTP and abilities of unlabeled nucleotide analogs to inhibit binding and to stimulate PLC in myo-[(3)H]inositol-loaded ampullas were determined. Specific [(3)H]UTP binding was competitively inhibited by UTP [apparent dissociation binding constant = 0.8 microM; Hill coefficient = 0.7]. Scatchard analysis revealed a minor class of high-affinity binding sites [45 fmol UTP bound/microgram protein; dissociation constant (K(D1)) = 0.4 microM] and a major class of moderate-affinity binding sites (365 fmol UTP bound/microgram protein; K(D2) = 10 microM). The stereospecificity pattern for UTP analog recognition was UMP > UDP >/= ADP = UTP = dTTP > adenosine 5'-O-(3-thiotriphosphate) = ATP = CTP = 2'-and 3'-O-4-(benzoylbenzoyl)-ATP (Bz-ATP) >/= AMP >/= 2-methylthio-ATP = alpha,beta-methylene-ATP > uridine = diadenosine tetraphosphate (Ap(4)A); cAMP and adenosine were inactive. Antagonist recognition pattern was DIDS = pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) = reactive blue 2 > suramin. The rank order of potencies for agonist-induced PLC activation was UDP >/= UTP >/= Ap(4)A >/= UMP = Bz-ATP; uridine was inactive. UTP-stimulated PLC activity was inhibited by DIDS = reactive blue 2 = PPADS > suramin. These results suggest that the population of [(3)H]UTP-labeled binding sites is heterogeneous, with a low number of high-affinity UTP receptors whose function(s) need to be determined and a large number of moderate-affinity receptors triggering PLC activation.     

Up4A stimulates endothelium-independent contraction of isolated rat pulmonary artery

Extracellular nucleotides, such as ATP, UDP, and UTP, regulate pulmonary vascular tone through P2X and P2Y receptors. Recently, uridine adenosine tetraphosphate (Up(4)A) was reported as a novel endothelium-derived vasoconstrictive factor. Up(4)A contains both purine and pyrimidine moieties, which potentially activate P2X and P2Y receptors. The present study examined the effect of Up(4)A on contractility of isolated rat pulmonary artery. Up(4)A at 1-100 microM stimulated contraction in a concentration-dependent manner. Up(4)A was equipotent as UTP and UDP in the endothelium-denuded artery while much more effective than UTP and UDP in endothelium-intact preparations. The vasoconstrictor effect of Up(4)A was inhibited by suramin but not IP(5)I or desensitization of P2X receptors with alpha,beta-methylene-ATP (alpha,beta-Me-ATP). Up(4)A-induced contraction was also inhibited by pretreatment with thapsigargin, nitrendipine, or EGTA but unaffected by H1152. Furthermore, unlike ATP and UTP, Up(4)A did not induce relaxation of endothelium-intact preparations precontracted with phenylephrine. These results suggest that Up(4)A is a potent vasoconstrictor, but not a vasodilator, of the rat pulmonary artery. Up(4)A likely acts through a suramin-sensitive P2Y receptor. The contractile effect of Up(4)A involves the entry of extracellular Ca(2+) and release of Ca(2+) from intracellular stores but not Ca(2+) sensitization via the RhoA/Rho kinase pathway. Up(4)A, therefore, potentially plays an important role in the regulation of pulmonary vascular tone.     

H(2)O(2) modulates purinergic-dependent calcium signalling in osteoblast-like cells

Reactive oxygen species (ROS) have long been considered as toxic by-products of aerobic metabolism and appear involved in the pathogenesis of degenerative diseases. The physiological role of ROS as second messengers in cell signal transduction is, on the other hand, increasingly recognized. Here we investigated the effects of H(2)O(2) and extracellular nucleotides on calcium signalling in four osteoblastic cell lines. In the highly differentiated HOBIT cells, sensitive to nanomolar concentrations of ADP and UTP, millimolar H(2)O(2) induced oscillatory increases of the cytosolic calcium concentration followed by a steady and sustained calcium increase. Long lasting rhythmic calcium activity was induced by micromolar H(2)O(2) doses. The H(2)O(2)-induced calcium signals, due to both release from intracellular stores and influx from the extracellular milieu, were totally prevented by incubating the cells with the P2 receptor antagonist suramin or with the ATP/ADP hydrolyzing enzyme apyrase. In the osteosarcoma SaOS-2 cells micromolar H(2)O(2) failed to evoke calcium signals and millimolar H(2)O(2) induced a slowly developing calcium influx which was unaffected by suramin and apyrase. These cells responded to micromolar concentrations of ATP and ADP, but were largely insensitive to UTP. ROS 17/2.8 osteosarcoma cells were totally insensitive to ATP, ADP and UTP in keeping with the evidence that these cells lack functional purinergic receptors. In these cells, H(2)O(2) up to 1mM did not increase the cytosolic calcium concentration. In ROS/P2Y(2) cells, stably expressing the P2Y(2) receptor, spontaneous calcium oscillations were observed in 38% of the population and nanomolar concentration of extracellular ATP or UTP activated oscillations in quiescent cells. Spontaneous calcium signals were inhibited by suramin and apyrase. In these cells H(2)O(2) induced oscillatory calcium activity that was blocked by suramin and apyrase. The sensitivity of ROS/P2Y(2) cells to UTP decreased significantly in the presence of DTT, which was effective also in inhibiting spontaneous calcium oscillations. On the other hand, the membrane-impermeant thiol oxidant DTNB induced calcium oscillations that were inhibited by incubating the cells with suramin or apyrase. Since peroxide did not increase extracellular ATP in these cell lines, we propose that, in osteoblasts, mild oxidative conditions could activate purinergic signalling through the sensitization of P2Y(2) receptor.     

Effect of purinergic agonists and antagonists on insulin secretion from INS-1 cells (insulinoma cell line) and rat pancreatic islets

The effects of purinergic agonists on insulin release are controversial in the literature. In our studies (mainly using INS-1 cells, but also using rat pancreatic islets), ATP had a dual effect on insulin release depending on the ATP concentration: increasing insulin release (EC50 approximately/= 0.0032 microM) and inhibiting insulin release (EC50 approximately/= 0.32 microM) at both 5.6 and 8.3 mM glucose. This is compatible with the view that either two different receptors are involved, or the cells desensitize and (or) the effect of an inhibitory degradation product such as adenosine (ectonucleotidase effect) emerges. The same dual effects of ATP on insulin release were obtained using rat pancreatic islets instead of INS-1 cells. ADPbetaS, which is less degradable than ATP and rather specific for P2Y1 receptors, had a dual effect on insulin release at 8.3 mM glucose: stimulatory (EC50 approximately/= 0.02 microM) and inhibitory (EC50 approximately/= 0.32 microM). The effectiveness of this compound indicates the possible involvement of a P2Y1 receptor. 2-Methylthio-ATP exhibited an insulinotropic effect at very high concentrations (EC50 approximately/= 15 microM at 8.3 mM glucose). This indicated that distinct P2X or the P2Y1 receptor may be involved in these insulin-secreting cells. UTP increased insulin release (EC50 approximately/= 2 microM) very weakly, indicating that a P2U receptor (P2X3 or possibly a P2Y2 or P2Y4) are not likely to be involved. Suramin (50 microM) antagonized the insulinotropic effect of ATP (0.01 microM) and UTP (0.32 microM). Since suramin is not selective, the data indicated that various P2X and P2Y receptors may be involved. PPADS (100 microM), a P2X and P2Y1,4,6 receptor antagonist, was ineffective using either low or high concentrations of ATP and ADPbetaS, which combined with the suramin data hints at a P2Y receptor effect of the compounds. Adenosine inhibited insulin release in a concentration-dependent manner. DPCPX (100 microM), an adenosine (A1) receptor antagonist, inhibited the inhibitory effects of both adenosine and of high concentrations of ATP. Adenosine deaminase (1 U/mL) abolished the inhibitory effect of high ATP concentrations, indicating the involvement of the degradation product adenosine. Repetitive addition of ATP did not desensitize the stimulatory effect of ATP. U-73122 (2 microM), a PLC inhibitor, abolished the ATP effect at low concentrations. The data indicate that ATP at low concentrations is effective via P2Y receptors and the PLC-system and not via P2X receptors; it inhibits insulin release at high concentrations by being metabolized to adenosine.     

P2Y-purinoceptor mediated inhibition of L-type Ca2+ channels in rat pancreatic beta-cells

We used the patch-clamp technique to study the effects of extracellular ATP on the activity of ion channels recorded in rat pancreatic beta-cells. In cell-attached membrane patches, action currents induced by 8.3 mM glucose were inhibited by 0.1 mM ATP, 0.1 mM ADP or 15 microM ADPbetaS but not by 0.1 mM AMP or 0.1 mM adenosine. In perforated membrane patches, action potentials were measured in current clamp, induced by 8.3 mM glucose, and were also inhibited by 0.1 mM ATP with a modest hyperpolarization to -43 mV. In whole-cell clamp experiments, ATP dose-dependently decreased the amplitudes of L-type Ca2+ channel currents (ICa) to 56.7+/-4.0% (p<0.001) of the control, but did not influence ATP-sensitive K+ channel currents observed in the presence of 0.1 mM ATP and 0.1 mM ADP in the pipette. Agonists of P2Y purinoceptors, 2-methylthio ATP (0.1 mM) or ADPbetaS (15 microM) mimicked the inhibitory effect of ATP on ICa, but PPADS (0.1 mM) and suramin (0.2 mM), antagonists of P2 purinoceptors, counteracted this effect. When we used 0.1 mM GTPgammaS in the pipette solution, ATP irreversibly reduced ICa to 58.4+/-6.6% of the control (p<0.001). In contrast, no inhibitory effect of ATP was observed when 0.2 mM GDPbetaS was used in the pipette solution. The use of either 20 mM BAPTA instead of 10 mM EGTA, or 0.1 mM compound 48/80, a blocker of phospholipase C (PLC), in the pipette solution abolished the inhibitory effect of ATP on ICa, but 1 microM staurosporine, a blocker of protein kinase C (PKC), did not. When the beta-cells were pretreated with 0.4 microM thapsigargin, an inhibitor of the endoplasmic reticulum (ER) Ca2+ pump, ATP lost the inhibitory effect on ICa. These results suggest that extracellular ATP inhibits action potentials by Ca2+-induced ICa inhibition in which an increase in cytosolic Ca2+ released from thapsigargin-sensitive store sites was brought about by a P2Y purinoceptor-coupled G-protein, PI-PLC and IP3 pathway.     

Synaptic mechanisms during re-innervation of rat arterial chemoreceptors in co-culture.

The carotid body plays a key role in the control of ventilation during hypoxia, a stimulus that releases catecholamines and other neurotransmitters from chemoreceptor (type 1) cells. Using co-cultures of rat type 1 clusters and 'juxtaposed' petrosal neurons (JPN), we recently showed that hypoxic chemotransmission is mediated via co-release of ACh and ATP. Recordings from JPN at functional, regenerated 'synapses' in vitro revealed spontaneous activity consisting of random e.p.s.p.'s and/or action potentials. This activity depended on chemical transmission since it was inhibited by extracellular solutions containing low Ca2+/high Mg2+, or blockers of nicotinic (e.g. 1-2 microM mecamylamine) and/or P2 purinergic (suramin or reactive blue 2; 10-50 microM) receptors. These solutions also inhibited hypoxia-evoked responses in JPN. The newly formed 'synapses' appeared stable, allowing repeated demonstration of hypoxic chemotransmission in the same JPN after at least a approximately 24-h re-incubation period. Immunofluorescence studies in situ revealed positive staining of P2X2 and P2X3 purinoceptor subunits in chemoafferent nerve terminals, but not type 1 cells; in contrast, both elements were immunopositive for the synaptic vesicle antigen (SV2). These data further support a co-transmitter role for ATP and the involvement of heteromeric P2X2/P2X3 purinoceptors in carotid body function.     

High reactivity of aortic fibroblasts to vasoactive agents: endothelins, bradykinin and nucleotides.

Cultured aortic fibroblasts express high affinity Et-1 binding sites that poorly discriminate between Et-1 and Et-3. Both endothelins activate phospholipase C hence indicating the presence of ETB receptors. Fibroblasts respond to bradykinin by large activations of phospholipase C and increases in [Ca2+]i in a manner that was abolished by D-Arg, [Hyp3,Thi5,8,D-Phe7]-bradykinin, thus indicating the presence of B2 kinin receptors. Finally, ATP, UTP and ADP increases [Ca2+]i in aortic fibroblasts via a nucleotide receptor that has a higher affinity for ATP and UTP (3 microM) than for ADP (50 microM) and that is distinct from P2x and P2y purinoceptors.     

Evidence for the presence of P2-purinoceptors at the surface of human articular chondrocytes in monolayer culture

Extracellular purines can act at purinoceptors to influence metabolic processes. Nucleotide-metabolizing ectoenzymes may modulate such purinergic effects, and their occurrence in a tissue may suggest the presence of purinoceptors. Thus, following the identification of ecto-nucleoside triphosphate pyrophosphatase in cultured human articular chondrocytes, we have studied whether these cells express P2-type purinoceptors. Release of prostaglandin E (PGE) was monitored, since articular chondrocytes synthesize and secrete PGE, and activation of P2-purinoceptors frequently results in enhanced prostaglandin production. Extracellular ATP and ADP stimulated PGE production, whereas AMP and adenosine had only limited effects. ATP concentrations as low as 5 microM were effective, and maximal responses were achieved at 50-100 microM ATP. GTP, UTP and ITP also elicited responses, but tended to be less effective than ATP at equivalent concentrations. Of the analogues of ATP that were tested, only adenosine 5'-(beta,gamma-methylene)triphosphate stimulated PGE production. The response to extracellular ATP was virtually abolished by indomethacin. Treatment of the cells with the P1-purinoceptor antagonist, 8-phenyltheophylline, or with pertussis toxin reduced both basal and ATP-stimulated PGE production, but did not substantially decrease the ratio of ATP-stimulated to basal PGE production. These results indicate the presence of P2-purinoceptors in cultured human articular chondrocytes, and suggest that extracellular ATP may have physiological and pathological effects in human articular cartilage.     

ATP in the mechanotransduction pathway of normal human chondrocytes

Extracellular nucleotides have been shown to have diverse effects on chondrocyte function, generally acting via P2 purinoceptors. We have previously shown that mechanical stimulation at 0.33 Hz of normal human chondrocyte cultures causes cellular hyperpolarisation, while chondrocytes derived from osteoarthritic (OA) cartilage depolarise. Experiments have been undertaken to establish whether ATP is involved in the response of the chondrocyte to mechanical stimulation. Chondrocytes, isolated from normal and OA cartilage obtained, with consent, from human knee joints following surgery, were cultured in non-confluent monolayer. Cells were mechanically stimulated at 0.33 Hz for 20 minutes at 37 degrees C in the presence or absence of inhibitors of ATP signalling, or were stimulated by the addition of exogenous ATP or derivatives, and electrophysiological measurements recorded. Samples of medium bathing the cells were collected before and after mechanical stimulation, and the concentration of ATP in the cell medium was measured. Total RNA was extracted from cultured chondrocytes, reverse-transcribed and used for RT-PCR with primers specific for P2Y2 purinoceptors. ATP, UTP 2-methylthioadenosine and alphabeta-methylene adenosine 5'-triphosphate all induced a hyperpolarisation response in normal human articular chondrocytes. No significant change was observed in the membrane potentials of chondrocytes isolated from OA cartilage following the addition of these nucleotides to the medium. In normal chondrocytes, the hyperpolarisation induced by ATP was blocked by the presence of apamin, indicating the involvement of small-conductance calcium-activated potassium channels. Following mechanical stimulation of normal chondrocytes, an increase was observed in ATP concentration in the cell culture medium bathing the cells. The presence within the culture medium of suramin or pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) prior to and during the period of mechanical stimulation abolished the hyperpolarisation response in normal chondrocytes. The presence of mRNA for P2Y2 purinoceptors was demonstrated in both normal and OA chondrocytes by RT-PCR. These results suggest that ATP has a role in the response of normal chondrocytes to mechanical stimulation, via P2Y2 purinoceptors. This response appears to be different in chondrocytes derived from OA cartilage, and may be important in the progression of this disease.     

Dual regulation of arachidonic acid release by P2U purinergic receptors in dibutyryl cyclic AMP-differentiated HL60 cells.

ATP promoted biphasic effects on both basal and fMLP-stimulated arachidonic acid (AA) release in neutrophil-like HL60 cells: stimulation in the micromolar range (EC50 = 3.2 +/- 0.9 microM) and inhibition at higher concentrations (EC50 = 90 +/- 11 microM). ATP also inhibited UTP- and platelet activating factor-stimulated AA release. Only stimulatory effects of ATP on basal or fMLP-stimulated phospholipase C were observed. The inhibitory effect of ATP on AA release was not due to reacylation of released AA, chelation of extracellular Ca2+, cell permeabilization, or changes in the rise of [Ca2+]i induced by agonist. The inhibition was rapid, being detected within 5-15 s. The inhibitory effect of ATP on fMLP-stimulated AA release could be desensitized by pretreatment of the cells with 2 mM ATP, but not 20 microM ATP, the concentration that resulted in maximal release of AA and inositol phosphates. The inhibition by ATP was neither dependent on generation of adenosine by ATP hydrolysis nor the result of direct interaction of ATP with P1 purinergic receptors. Among other nucleotides tested (CTP, GTP, ITP, TTP, XTP, adenosine 5'-(beta,gamma-methylene)triphosphate (AMP-PCP), adenyl-5'-yl imidodiphosphate (AMP-P(NH)P), ADP, adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), and UTP), only UTP and ATP gamma S displayed biphasic effects with potencies and efficacies almost identical to those of ATP. The other nucleotides only exhibited stimulatory effects (EC50 = 60-300 microM). The results are consistent with a model of dual regulation of AA release by two distinct subtypes of P2U receptors in HL60 cells.     

P2Y receptor-mediated inhibition of tumor necrosis factor alpha -stimulated stress-activated protein kinase activity in EAhy926 endothelial cells

In the EAhy926 endothelial cell line, UTP, ATP, and forskolin, but not UDP and epidermal growth factor, inhibited tumor necrosis factor alpha (TNFalpha)- and sorbitol stimulation of the stress-activated protein kinases, JNK, and p38 mitogen-activated protein (MAP) kinase, and MAPKAP kinase-2, the downstream target of p38 MAP kinase. In NCT2544 keratinocytes, UTP and a proteinase-activated receptor-2 agonist caused similar inhibition, but in 13121N1 cells, transfected with the human P2Y(2) or P2Y(4) receptor, UTP stimulated JNK and p38 MAP kinase activities. This suggests that the effects mediated by P2Y receptors are cell-specific. The inhibitory effects of UTP were not due to induction of MAP kinase phosphatase-1, but were manifest upstream in the pathway at the level of MEK-4. The inhibitory effect of UTP was insensitive to the MEK-1 inhibitor PD 098059, changes in intracellular Ca(2+) levels, or pertussis toxin. Acute phorbol 12-myristate 13-acetate pretreatment also inhibited TNFalpha-stimulated SAP kinase activity, while chronic pretreatment reversed the effects of UTP. Furthermore, the protein kinase C inhibitors Ro318220 and Go6983 reversed the inhibitory action of UTP, but GF109203X was ineffective. These results indicate a novel mechanism of cross-talk regulation between P2Y receptors and TNFalpha-stimulated SAP kinase pathways in endothelial cells, mediated by Ca(2+)-independent isoforms of protein kinase C.     

Effects of ATP on intracellular calcium dynamics of neurons and satellite cells in rat superior cervical ganglia

Adenosine 5'-triphosphate (ATP) which is released from neuronal and non-neuronal tissues interacts with cell surface receptors to produce a broad range of physiological responses. The present study addressed the issue of whether the cells of the superior cervical ganglia (SCG) respond to ATP. To this end, the dynamics of the intracellular calcium ion concentration ([Ca2+]i) of neurons and satellite cells in intact SCG was analyzed by laser scanning confocal microscopy. ATP produced an increase of [Ca2+]i in both neurons and satellite cells; initially, ATP elicited [Ca2+]i increase in satellite cells and, subsequently, a [Ca2+]i change in neurons was observed. P1 purinoceptor agonists had no effect on this process, but P2 purinoceptor agonists induced [Ca2+]i increase and suramin totally inhibited ATP-induced [Ca2+]i dynamics in both neurons and satellite cells. In satellite cells, Ca2+ channel blockers and the removal of extracellular Ca2+, but not thapsigargin pretreatment, abolished ATP-induced [Ca2+]i dynamics. In contrast, thapsigargin pretreatment abolished ATP-induced [Ca2+]i dynamics in neurons. Reactive blue-2 inhibited the ATP-induced reaction on neurons alone. Uridine 5'-triphosphate caused a [Ca2+]i increase in neurons and alpha,beta-methylene ATP caused a [Ca2+]i increase in satellite cells. We concluded that neurons respond to extracellular ATP mainly via P2Y purinoceptors and that satellite cells respond via P2X purinoceptors.     

Effects of suramin, an anti-human immunodeficiency virus reverse transcriptase agent, on protein kinase C. Differential activation and inhibition of protein kinase C isozymes.

Suramin inhibited protein kinase C (PKC) type I-III activity in a concentration-dependent manner. Similar inhibitory effects were observed with M-kinase, the constitutively active catalytic fragment of PKC, and autophosphorylation of PKC types I-III. Kinetic experiments indicated that suramin competitively inhibits activity with respect to ATP (Ki = 17, 27, and 31 microM, respectively) and that it can also inhibit by interaction with the substrate histone III-S. With protamine as the Pi acceptor, suramin inhibition was dependent on lipid, being approximately 4-fold less sensitive to inhibition in the absence of phosphatidylserine and diacylglycerol than in their presence. Suramin at low concentrations (10-40 microM), in the presence of Ca2+ and absence of lipid, was able to stimulate kinase activity (approximately 200-400%) in a type-dependent manner and at higher concentrations inhibited activity with histone III-S as substrate. These results indicate that suramin, a hexa-anionic hydrophobic compound, can act as a negatively charged phospholipid analog in activating PKC in the presence of Ca2+ and absence of lipid and can inhibit Ca2+/phosphatidylserine/diacylglycerol-stimulated kinase activity at higher concentrations by competing with ATP or by interaction with the exogenous substrate. Suramin inhibited cAMP-dependent protein kinase much less potently (IC50 = 656 microM) than PKC. The ability of suramin to inhibit PKC-mediated processes in intact cells was tested using the phorbol ester-stimulated respiratory burst of neutrophils as a model system. The respiratory burst of human neutrophils, when preincubated with suramin and then stimulated with phorbol ester, was inhibited in a concentration-dependent manner, suggesting that suramin may also be able to inhibit PKC-mediated processes in intact cells.     

Suramin prevents import of acyl-CoA oxidase into rat liver peroxisomes.

The inhibitory effect of suramin on the import of [35S]acyl-CoA oxidase into purified rat liver peroxisomes was investigated in vitro. The import of acyl-CoA oxidase was inhibited completely by 10 microM suramin, whilst the latency of catalase remained unchanged. The important value decreased 60% by pretreatment of peroxisomes with 10 microM suramin, but it did not decrease by pretreatment of translation products. Polysulfonate compounds which have two clusters of negative charges, such as Cibacron blue F3GA and Trypan blue, as well as suramin, inhibited the import, whilst mono- and disulfonate compounds did not.