Co-existence of P2Y-and PPADS-insensitive P2U-purinoceptors in endothelial cells from adrenal medulla

1. We have studied the effects of purinoceptor stimulation on Ca2+ signals in bovine adrenomedullary endothelial cells. [Ca2+]i was determined with the fluorescent probe fura-2 both in population samples and in single, isolated, endothelial cells in primary culture and after subculturing. 2. In endothelial cells, maintained in culture for more than one passage, several purinoceptor agonists elicited clear [Ca2+]i transient peaks that remained in the absence of extracellular Ca2+. Adenosine 5'-triphosphate (ATP) and uridine 5'-triphosphate (UTP) were equipotently active, with EC50 values of 8.5 +/- 0.9 microM and 6.9 +/- 1.5 microM, respectively, whereas 2-methylthioadenosine 5'-triphosphate (2MeSATP), adenosine 5'-(alpha, beta-methylene)triphosphate (alpha, beta-MeATP) and adenosine(5')tetraphospho(5')adenosine (Ap4A) were basically inactive. Adenosine 5'-O-(2-thiodiphosphate) (ADP beta S) was a weak agonist. The apparent potency order was UTP = ATP > ADP beta S > 2MeSATP > alpha, beta-MeATP. 3. Cross-desensitization experiments revealed that UTP or ATP, added sequentially at concentrations of maximal effect, could completely abolish the [Ca2+]i response to the second agonist. ADP beta S exerted only a partial desensitization of the response to maximal ATP, in accordance with its lower potency in raising [Ca2+]i. 4. The effect on [Ca2+]i of 100 microM ATP in subcultured cells was reduced by only 25% with 100 microM suramin pretreatment and was negligibly affected by exposure to 10 microM pyridoxalphosphate-6-azophenyl-2', 4'-disulphonic acid (PPADS). The concentration-effect curve for ATP was not significantly affected by PPADS, but was displaced to the right by a factor of 6.5 by 100 microM suramin. 5. In primary cultures, clear [Ca2+]i responses were elicited by 2MeSATP. Suramin totally and selectively blocked 2MeSATP responses, whereas UTP-evoked [Ca2+]i transients were mainly unaffected by suramin or PPADS. Over 80% of cells tested showed responses to both 2MeSATP and UTP. The [Ca2+]i response to UTP was not desensitized in the presence of 2MeSATP. 6. ATP and UTP stimulated the release of preloaded [3H]-arachidonic acid ([3H]-AA), both in the presence and in the absence of extracellular Ca2+, by approximately 135% with respect to basal levels. Suramin and PPADS enhanced, rather than inhibited, the [3H]-AA releasing effect of ATP by 2.5 times. Suramin also potentiated the effect of the calcium ionophore A23187. 7. These results indicate that endothelial cells from adrenomedullary capillaries co-express both P2Y- and P2U-purinoceptors. P2Y-purinoceptors are lost in culture with the first passage of the cells. The P2U-purinoceptor subtype present in these cells is insensitive to PPADS and thus similar to that found in aortic endothelial cells.     

Ca2+ mobilization in bovine corneal endothelial cells by P2 purinergic receptors

PURPOSE: To characterize Ca2+ mobilization by P2 receptors in the bovine corneal endothelial cells (BCEC). METHODS: Changes in intracellular Ca2+ ([Ca2+]i) were measured by fluorescence imaging of cultured and fresh BCEC cells loaded with the Ca2+-sensitive dye Fura-PE3. Relative rates of Ca2+ influx were measured employing Mn2+ as a surrogate for Ca2+. RESULTS: Exposure of cultured cells to uridine 5'-triphosphate (UTP), 2-methyl-thio ATP (msATP) and ATP caused biphasic changes in [Ca2+]i consisting of a peak followed by a plateau phase. Based on the peak responses to 100 microM agonist, the magnitude of UTP responses were similar to that of ATP but greater than that of msATP or ADP. UTP and msATP stimulated Mn2+ influx following [Ca2+]i peak similar to that observed in response to cyclopiazonic acid (CPA), an inhibitor of ER Ca2+-ATPase. Under Ca2+-free conditions, peak responses were similar to those in the presence of external Ca2+, but reduced when the cells were pre-exposed to CPA. Reactive Blue-2 (RB2), inhibited msATP responses by 60.4 +/- 18.8% but UTP responses by only 10.6 +/- 9.5%. Repeated exposures to UTP or msATP reduced [Ca2+]i mobilization indicating homologous desensitization. Response to UTP was not affected by a prior exposure to msATP. However, response to msATP was reduced by a prior exposure to UTP indicating mixed heterologous desensitization. Fresh cells responded to UTP (50 microM) with temporal characteristics of [Ca2+]i mobilization similar to that of cultured cells. CONCLUSION: BCEC express P2 receptors belonging to the P2Y subfamily. The emptying of the IP3-sensitive stores, leading to the initial peak in [Ca2+]i response, subsequently caused capacitative Ca2+ influx leading to the onset of the plateau phase. A significant homologous desensitization to UTP and msATP, selective heterologous desensitization between UTP and msATP, and selective inhibition by RB2 indicate the coexistence of multiple P2Y receptors.     

P2 receptor-mediated signal transduction in Ehrlich ascites tumor cells

The mechanisms, by which the P2 receptor agonists adenosine 5'-triphosphate (ATP) and uridine 5'-triphosphate (UTP) evoke an increase in the free cytosolic calcium concentration ([Ca2+]i) and in intracellular pH (pHi), have been investigated in Ehrlich ascites tumor cells. The increase in [Ca2+]i evoked by ATP or UTP is abolished after depletion of intracellular Ca2+ stores with thapsigargin in Ca2+-free medium, and is inhibited by U73122, an inhibitor of phospholipase C (PLC), indicating that the increase in [Ca2+]i is primarily due to release from intracellular, Ins(1,4,5)P3-sensitive Ca2+ stores. ATP also activates a capacitative Ca2+-entry pathway. ATP as well as UTP evokes a biphasic change in pHi, consisting of an initial acidification followed by alkalinization. Suramin and 4,4'-diisothiocyano-2,2'-stilbene-disulfonic acid (DIDS) inhibit the biphasic change in pHi, apparently by acting as antagonists at P2 receptors. The alkalinization evoked by the P2 receptor agonists is found to be due to activation of a 5'-(N-ethyl-N-isopropyl)amiloride (EIPA)-sensitive Na+/H+ exchanger. ATP and UTP elicit rapid cell shrinkage, presumably due to activation of Ca2+ sensitive K+ and Cl- efflux pathways. Preventing cell shrinkage, either by incubating the cells at high extracellular K+ concentration, or by adding the K+-channel blocker, charybdotoxin, does not affect the increase in [Ca2+]i, but abolishes the activation of the Na+/H+ exchanger, indicating that activation of the Na+/H+ exchanger is secondary to the Ca2+-induced cell shrinkage.     

Dual regulation of cerebrovascular tone by UTP: P2U receptor-mediated contraction and endothelium-dependent relaxation

1. The mechanisms of vascular tone regulation by extracellular uridine 5'-triphosphate (UTP) were investigated in bovine middle cerebral arterial strips. Changes in cytosolic Ca2+ concentration ([Ca2+]i) and force were simultaneously monitored by use of front-surface fluorometry of fura-2. 2. In the arterial strips without endothelium, UTP (0.1 microM-1 mM) induced contraction in a concentration-dependent manner. However, when the endothelium was kept intact, cumulative application of UTP (0.1-100 microM) (and only at 1 mM) induced a modest phasic contraction in arterial strips. This endothelium-dependent reduction of the UTP-induced contraction was abolished by 100 microM N omega-nitro-L-arginine (L-NOARG) but not by 10 microM indomethacin. In the presence of intact endothelium, UTP (30 microM) induced a transient relaxation of the strips precontracted with 30 nM U-46619 (a stable analogue of thromboxane A2), which was completely inhibited by pretreatment with L-NOARG but not with indomethacin. 3. In the endothelium-denuded strips, the contractile response to UTP was abolished by desensitization to either ATP gamma S or ATP (P2U receptor agonists), but not by desensitization to alpha, beta-methylene-ATP (P2x receptor agonist) or to 2-methylthio-ATP (P2Y receptor agonist). Desensitization to UTP abolished the contractile response to ATP. 4. In the endothelium-denuded artery, a single dose application of UTP induced an initial transient, and subsequently lower but sustained increase in [Ca2+]i and force. In the absence of extracellular Ca2+, UTP induced only the initial transient increases in [Ca2+]i and force, while the sustained increases in [Ca2+]i and force were abolished. UTP (1 mM) had no effect on the basic [Ca2+]i-force relationship obtained on cumulative application of extracellular Ca2+ at steady state of 118 mM K(+)-depolarization-induced contraction. 5. We conclude that in the presence of an intact endothelium, UTP-induced relaxation of preconstricted middle cerebral artery is mainly mediated indirectly, by the production of an endothelium-derived relaxing factor, but at high doses of UTP, vascular smooth muscle contraction is mediated directly via activation of P2U purinoceptor and [Ca2+]i elevation without Ca(2+)-sensitization of the contractile apparatus. UTP may thus exert a dual regulatory effect upon cerebrovascular tone, but in cases where the endothelium is impaired, it may also act as a significant vasoconstrictor.     

The regulation of aortic endothelial cells by purines and pyrimidines involves co-existing P2y-purinoceptors and nucleotide receptors linked to phospholipase C

1. We have examined the phospholipase C responses in bovine aortic endothelial cells to purines (ATP, ADP and analogues) and the pyrimidine, uridine triphosphate (UTP). 2. The cells responded to purines in a manner consistent with the presence of P2y purinoceptors; both 2-methylthioadenosine 5'-triphosphate (2MeSATP) and adenosine 5'-0-(2-thiodiphosphate) (ADP beta S) were potent agonists (EC50 0.41 microM and 0.85 microM respectively) while beta, gamma-methylene ATP at 300 microM was not. 3. The cells also responded to UTP. The maximal response to UTP was less than that for either 2MeSATP and ADP beta S while adenosine 5'-0-(3-thiotriphosphate) (ATP gamma S) gave the largest maximal response. 4. The concentration-effect curve to UTP was additive in the presence of either 2MeSATP or ADP beta S. However, the concentration-effect curves to ATP gamma S reached the same maximum in the presence or absence of UTP. 5. Suramin, at concentrations between 10 microM and 100 microM was a competitive antagonist for the response to ADP beta S and 2MeSATP but not the response to UTP. 6. The results show that there are two separate, co-existing, receptor populations: P2y-purinoceptors (responding to purines) and nucleotide receptors (responding to both purines and pyrimidines). We conclude that purines such as ATP/ADP may regulate aortic endothelial cells by interacting with two phospholipase C-linked receptors.     

Differential regulation of inositol 1,4,5-trisphosphate by co-existing P2Y-purinoceptors and nucleotide receptors on bovine aortic endothelial cells

1. We have examined the inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) responses in bovine aortic endothelial (BAE) cells to purines (ATP, ADP and analogues) and the pyrimidine, uridine triphosphate (UTP). 2. Exchange of medium on BAE cells in the absence of agonist was found to be a stimulus for Ins(1,4,5)P3 generation. BAE cells stimulated with 100 microM ATP, 30 microM 2MeSATP (an agonist at P2Y-purinoceptors but not nucleotide receptors) or 100 microM UTP (an agonist at nucleotide receptors but not P2Y-purinoceptors) gave Ins(1,4,5)P3 responses above that caused by exchange of medium. The time course was rapid, with peak response within the first 5 s and levels returning close to basal after 30 s of stimulation. 3. Significant differences in Ins(1,4,5)P3 responses to 100 microM UTP and 30 microM 2MeSATP stimulation were observed. The response to UTP was reproducibly more sustained than that to 2MeSATP. 4. Stimulation of BAE cells with 100 microM UTP plus 30 microM 2MeSATP produced a response statistically indistinguishable from that predicted by addition of the responses to the two agonists in isolation. 5. The Ins(1,4,5)P3 response to UTP was attenuated to 25% of control by pretreatment of BAE cells with pertussis toxin. Responses to 2MeSATP and ADP were essentially unaffected. ATP stimulation was reduced to 65% of control. 6. Activation of protein kinase C with tetradecanoyl phorbol acetate (TPA) profoundly inhibited Ins(1,4,5)P3 responses to 2MeSATP and ADP but had no effect on UTP stimulation. The protein kinase C inhibitor, Ro 31-8220, enhanced responses to 2MeSATP, ADP and ATP but no effect was observed on UTP stimulation. 7. These observations show that nucleotide and P2Y-receptors mobilise the second messenger Ins(1,4,5)P3 by separate routes resulting in different patterns of generation and suggest that while ATP activates both receptors, ADP principally influences these cells by interacting with the P2Y-purinoceptors.     

ATP stimulation of Ca2+ -dependent plasminogen release from cultured microglia

1. ATP (10-100 microM), but not glutamate (100 microM), stimulated the release of plasminogen from microglia in a concentration-dependent manner during a 10 min stimulation. However, neither ATP (100 microM) nor glutamate (100 microM) stimulated the release of NO. A one hour pretreatment with BAPTA-AM (200 microM), which is metabolized in the cytosol to BAPTA (an intracellular Ca2+ chelator), completely inhibited the plasminogen release evoked by ATP (100 microM). The Ca2+ ionophore A23187 induced plasminogen release in a concentration-dependent manner (0.3 microM to 10 microM). 2. ATP induced a transient increase in the intracellular calcium concentration ([Ca2+]i) in a concentration-dependent manner which was very similar to the ATP-evoked plasminogen release, whereas glutamate (100 microM) had no effect on [Ca2+]i (70 out of 70 cells) in microglial cells. A second application of ATP (100 microM) stimulated an increase in [Ca2+]i similar to that of the first application (21 out of 21 cells). 3. The ATP-evoked increase in [Ca2+]i was totally dependent on extracellular Ca2+, 2-Methylthio ATP was active (7 out of 7 cells), but alpha,beta-methylene ATP was inactive (7 out of 7 cells) at inducing an increase in [Ca2+]i. Suramin (100 microM) was shown not to inhibit the ATP-evoked increase in [Ca2+]i (20 out of 20 cells). 2'- and 3'-O-(4-Benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP), a selective agonist of P2X7 receptors, evoked a long-lasting increase in [Ca2+]i even at 1 microM, a concentration at which ATP did not evoke the increase. One hour pretreatment with adenosine 5'-triphosphate-2', 3'-dialdehyde (oxidized ATP, 100 microM), a selective antagonist of P2X7 receptors, blocked the increase in [Ca2+]i induced by ATP (10 and 100 microM). 4. These data suggest that ATP may transit information from neurones to microglia, resulting in an increase in [Ca2+]i via the ionotropic P2X7 receptor which stimulates the release of plasminogen from the microglia.     

Anxiogenic-like consequences in animal models of complex partial seizures

Extracellular adenosine triphosphate (ATP) plays an important role in the regulation of endothelial function. However, its receptors and their signal-transduction pathways in major cerebral arterial endothelial cells are largely unknown. This study was undertaken functionally to classify the P2 purinoceptors in cultured bovine middle cerebral artery endothelial cells by using [Ca2+]i microfluorimetry. The rank order of potency to increase [Ca2+]i was 2-methylthio-ATP approximately ATP approximately uridine triphosphate (UTP) > adenosine diphosphate (ADP) > adenosine monophosphate (AMP) > alpha,beta-methylene-ATP > adenosine, suggesting that the effect was mediated by both P2y and P2u receptors. ATP, 2-methylthio-ATP, and UTP mobilized Ca2+ from intracellular stores and triggered Ca2+ entry. The effects of ATP, 2-methylthio-ATP, and UTP were reduced by phospholipase C inhibitor 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (NCDC), but only the effects of ATP and UTP were attenuated by pertussis toxin, indicating that P2y and P2u receptors may activate the same effector mechanisms by coupling to different G proteins. The [Ca2+]i entry caused by UTP was significantly reduced by the receptor-regulated Ca2+ channel blocker SK&F 96365, by P-450 inhibitor econazole and by inorganic Ca2+ entry blocker lanthanum. P2-receptor antagonists suramin, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), and reactive blue 2 reduced the effects of ATP and 2-methylthio-ATP, but not those of UTP, in a concentration-dependent manner. These studies suggest a coexistence of P2y and P2u receptors in cultured bovine middle cerebral artery endothelial cells.     

Long-term activation of capacitative Ca2+ entry in mouse microglial cells

The cytoplasmic free calcium concentration ([Ca2+]i) was measured in cultured microglial cells with the Ca2+-sensitive fluorescent dye Fura-2 using a digital imaging system. Stimulation of P2 purinergic receptors by ATP or UTP always evoked a [Ca2+]i elevation. The ATP-induced Ca2+ response involved both Ca2+ influx through ionotropic receptors and Ca2+ release from intracellular pools, whereas UTP selectively stimulated intracellular Ca2+ release. When intracellular Ca2+ release was stimulated in the absence of extracellular Ca2+, the readmission of extracellular Ca2+ caused a large rebound [Ca2+]i increase. Following this rebound, [Ca2+]i did not return to the initial resting level, but remained for long periods of time (up to 20 min), at a new, higher steady-state level. Both the amplitude of the rebound Ca2+ transient and the new plateau level strongly correlated with the degree of intracellular Ca2+ depletion, indicating the activation of a store-operated Ca2+ entry pathway. The elevated steady-state [Ca2+]i level was associated with a significant increase in the plasma membrane permeability to Ca2+, as changes in extracellular Ca2+ were reflected in almost immediate changes of [Ca2+]i. Similarly, blocking plasma-lemmal Ca2+ channels with the non-specific agonist La3+ (50 microM) caused a decrease in [Ca2+]i, despite the continuous presence of Ca2+ ions in the extracellular medium. After the establishment of the new, elevated steady-state [Ca2+]i level, stimulation of P2U metabotropic purinoreceptors did not induce a [Ca2+]i response. In addition, application of either thapsigargin (1 microM) or carbonyl cyanide chlorophenyl hydrazone (10 microM) failed to affect [Ca2+]i. We conclude that the maximal depletion of intracellular Ca2+ stores in mouse brain microglia determines the long-term activation of a plasma membrane Ca2+ entry pathway. This activation appears to be associated with a significant decrease in the capability of the intracellular Ca2+ stores to take up cytosolic Ca2+ once they have been maximally depleted.     

Characterization of Ca2+ influx through recombinant P2X receptor in C6BU-1 cells

1. The effects of exogenous adenosine 5'-triphosphate (ATP) and alpha,beta-methylene ATP (alpha,beta meATP) on C6BU-1 cells transfected with P2X2 and P2X3 subtypes, separately or together (P2X2+3), were investigated using fura-2 fluorescence recording and whole-cell patch clamp recording methods. 2. Untransfected C6BU-1 cells showed no intracellular Ca2+ ([Ca2+]i) increase in response to depolarizing stimulation with high K+ or stimulation with ATP. There was no current induced by ATP under voltage clamp conditions in untransfected C6BU-1 cells. ATP caused Ca2+ influx only from extracellular sources in C6BU-1 cells transfected with the P2X subtypes, suggesting that the C6BU-1 cell line is suitable for the characterization of Ca2+ influx through the P2X subtypes. 3. In C6BU-1 cells transfected with the P2X2 subtype, ATP (more than 10 microM) but not alpha,beta meATP (up to 100 microM) evoked a rise in [Ca2+]i. 4. In the cells transfected with the P2X3 subtype, current responses under voltage clamp conditions were observed at ATP concentrations higher than 0.1 microM of alpha,beta meATP were required. This discrepancy in the concentration dependence of the agonist responses with respect to the [Ca2+]i rise and the current response was seen only with the P2X3 subtype. In addition, the agonist-induced rise in [Ca2+]i was observed only after the first application because of desensitization of this subtype. 5. In C6BU-1 cells co-transfected with P2X2 and P2X3, ATP at 1 microM evoked a [Ca2+]i rise. This responsiveness was higher than that of the other subtype combinations tested. The efficiency of expression was improved by co-transfection with P2X2 and P2X3, when compared to transfection with the P2X3 subtype alone. The desensitization of the P2X2+3 was apparently slower than that of the P2X3 subtype alone. Therefore, this combination could respond to the repeated application of agonists each time with a [Ca2+]i rise. 6. These results suggest that the P2X2 and P2X3 subtypes assemble a heteromultimer and that this heterogeneous expression acquires more effective Ca2+ dynamics than that by homogeneously expressed P2X2 or P2X3.     

Expression of functional P2-purinergic receptors in primary cultures of human colorectal carcinoma cells

Primary cell cultures of human colorectal carcinomas were established and characterized immunocytochemically. In the isolated cancer cells intracellular Ca2+ concentrations ([Ca2+]i) were measured by the fura-2 method. Stimulation with either extracellular ATP or UTP caused a biphasic rise of [Ca2+]i in a dose-dependent manner and cross-desensitization between both nucleotides was observed. The rank order of potency was ATP >== UTP > ATP-gamma-S > ADP > adenosine which is characteristic for a P2U-receptor subtype. Selective agonists of P1-, or P2X- purinoceptors had no effect on [Ca2+]i. The initial rise in [Ca2+]i was independent of extracellular calcium [Ca2+]e, whereas the second phase was not observed under [Ca2+]e-free conditions suggesting a capacitative Ca2+-entry-mechanism. Intracellular Ca2+ mobilization was proven by use of the Ca2+-ATPase inhibitor thapsigargin. P2U-specific mRNA could be detected by RT-PCR in both colorectal tumor tissues and in the human colorectal cancer cell line HT 29. In HT 29 cells, the hydrolysis-resistant ATP analog ATP-gamma-S inhibited cell proliferation and, also, induced apoptosis in a dose-dependent manner. Thus, human colorectal cancer cells express functional P2U-receptors which may play a role in the regulation of cell proliferation and apoptosis.     

Diverse signal transduction pathways mediated by endogenous P2 receptors in cultured rat cerebral cortical neurons

The present study was conducted to assess the intracellular signaling pathways mediated by receptors for ATP, uridine triphosphate (UTP), and 2-methylthio ATP (2-MeSATP), by monitoring patch-clamp currents and intracellular calcium mobilization in cultured rat cortical cerebral neurons. All three agonists evoked potassium currents and increased the intracellular free Ca2+ concentration ([Ca2+]i), and these effects were inhibited by the broad G-protein inhibitor guanosine-5'-O-(2-thiodiphosphate) (GDPbetaS) but not by the Gi/o-protein inhibitor pertussis toxin (PTX). UTP-evoked currents were inhibited by either the phospholipase C inhibitor neomycin or the selective protein kinase C (PKC) inhibitor GF109203X, and the rise in cytosolic Ca2+ was inhibited by either neomycin or the inositol 1,4,5-trisphosphate (IP3) receptor antagonist heparin, indicating that the UTP receptor involved phospholipase C-mediated phosphatidylinositol signaling. In contrast, 2-MeSATP-induced currents and rise in cytosolic Ca2+ were not inhibited by either neomycin, or GF109203X, or heparin. 2-MeSATP elicited single-channel currents in the cell-attached patch-clamp configuration and also in excised patches. The G-protein activator GTP gamma S induced single-channel currents in a fashion that mimicked the effect of 2-MeSATP. These data suggest that 2 MeSATP activated potassium channels by a direct action of G-protein beta gamma subunits and increased [Ca2+]i by a mechanism independent of phospholipase C stimulation and IP3 production. ATP-evoked currents were partially inhibited by either neomycin or GF109203X, although the rise in cytosolic Ca2+ was not affected by these inhibitors. ATP produced single-channel currents with two major classes of the slope conductance (86 and 95 pS) in cell-attached patches, each of which is consistent with that achieved by 2-MeSATP (85 pS) or UTP (96 pS); the currents with the lower conductance were observed in the outside-out patch-clamp configuration. These results indicate that P2 receptors for UTP and 2-MeSATP are linked to a PTX-insensitive G-protein involving different signal transduction pathways and that ATP responses are mediated by both of these P2 receptors.     

The effects of purine compounds on the isolated aorta of the frog Rana temporaria

1. In the isolated aorta of the frog, Rana temporaria, adenosine concentration-dependently, endothelium-independently relaxed adrenaline pre-constricted vessels. None of the adenosine analogues including D-5'-(N-ethylcarboxamide) adenosine (NECA), R- and S-N6-(2-phenylisopropyl) adenosine (R-and S-PIA) and 2-chloroadenosine (2-CA), or the more selective A1, A2 and A3 agonists cyclopentyladenosine (CPA), CGS 21680 and N6-(3-iodobenzyl) adenosine-5'-N-methylcarboxamide (IB-MECA) respectively, had any effect. 2. The non-selective adenosine antagonist, 8-p-sulphophenyl-theophylline (8-pSPT; 30 microM) failed to inhibit adenosine relaxations, as did NG-nitro-L-arginine methyl ester (L-NAME; 0.1 mM) and indomethacin (30 microM). 3. Adenosine 5'-triphosphate (ATP), alpha, beta-methylene ATP (alpha, beta-MeATP), beta, gamma-methylene ATP (beta, gamma-MeATP), 2-methylthio ATP (2-MeSATP) and uridine 5'-triphosphate (UTP) all concentration-dependently contracted the frog aorta. ATP and alpha, beta-MeATP were equipotent and more potent than UTP and beta, gamma-MeATP; 2-MeSATP had little activity. 4. The P2-purinoceptor antagonist, suramin (0.1 mM) inhibited contractions to alpha, beta-MeATP but not to ATP. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS; 30 microM) also inhibited contractions to alpha, beta-MeATP but not to ATP. Contractions to ATP were, however, inhibited by indomethacin (30 microM). 5. In conclusion, in the frog aorta there appears to be a novel subclass of P1-purinoceptor mediating vasodilatation, although like the A3 subclass it is not blocked by methylxanthines; a P2-purinoceptor mediates vasconstriction which resembles a P2x subtype, based on the agonist potency of alpha, beta-MeATP being more potent than 2-MeSATP (UTP has moderate activity) and PPADS is an effective antagonist. There is no evidence for the presence of a P2y-purinoceptor, mediating vasodilatation, in this preparation.     

Mechanical stress induces release of ATP from Ehrlich ascites tumor cells

The supernatant from a suspension of Ehrlich cells exposed to centrifugation at 700xg for 45 s induced a transient increase in the intracellular concentration of free, cytosolic Ca2+, [Ca2+]i, as well as activation of an outwardly rectifying whole-cell current when added to a suspension of non-stimulated cells. These effects were inhibited by suramin, a non-specific P2 receptor antagonist, and mimicked by ATP. Reversed phase HPLC analysis revealed that the supernatant from Ehrlich cells exposed to centrifugation contained 2. 6+/-0.2 microM ATP, and that the mechanical stress-induced release of ATP was inhibited by glibenclamide and verapamil, non-specific inhibitors of the cystic fibrosis transmembrane conductance regulator and P-glycoprotein, respectively. After trypan blue staining, less than 0.5% of the cells were unable to extrude the dye. Addition of extracellular ATP induced a suramin-sensitive, transient, concentration-dependent increase in [Ca2+]i, activation of an outwardly rectifying whole-cell current and a hyperpolarization of the plasma membrane. The ATP-induced hyperpolarization of the plasma membrane was strongly inhibited in the presence of charybdotoxin (ChTX), an inhibitor of several Ca2+-activated K+ channels, suggesting that stimulation of P2 receptors in Ehrlich cells evokes a Ca2+-activated K+ current. The relative potencies of several nucleotides (ATP, UTP, ADP, 2-MeSATP, alpha,beta-MeATP, bzATP) in eliciting an increase in [Ca2+]i, as well as the effect of repetitive addition of nucleotides were investigated. The results lead us to conclude that mechanical stimulation of Ehrlich cells leads to release of ATP, which in turn stimulates both P2Y1 and P2Y2 receptors, resulting in Ca2+ influx as well as release and activation of an outwardly rectifying whole-cell current.     

Characterization and localization of P2 receptors in rat submandibular gland acinar and duct cells

[Ca2+]i and the Cl- current were measured in isolated submandibular gland acinar and duct cells to characterize and localize the purinergic receptors expressed in these cells. In both cell types 2'-3'-benzoylbenzoyl (Bz)-ATP and ATP increased [Ca2+]i mainly by activation of Ca2+ influx. UTP had only minimal effect on [Ca2+]i at concentrations between 0.1 and 1 mM. However, a whole cell current recording showed that all nucleotides effectively activated Cl- currents. Inhibition of signal transduction through G proteins by guanyl-5'-beta-thiophosphate revealed that the effect of ATP on Cl- current was mediated in part by activation of a G protein-coupled and in part by a G protein-independent receptor. BzATP activated exclusively the G protein-independent portion, whereas UTP activated only the G protein-dependent portion of the Cl- current. Measurement of [Ca2+]i in the microperfused duct showed that ATP stimulated a [Ca2+]i increase when applied to the luminal or the basolateral sides. BzATP increased [Ca2+]i only when applied to the luminal side, whereas UTP at 100 microM increased -Ca2+-i only when applied to the basolateral side. The combined results suggest that duct and possibly acinar cells express P2z receptors in the luminal and P2u receptors in the basolateral membrane.     

Homologous and heterologous desensitisation of somatostatin-induced increases in intracellular Ca2+ and inositol 1,4,5-trisphosphate in CHO-K1 cells expressing human recombinant somatostatin sst5 receptors

The mechanisms responsible for somatostatin (SRIF)-induced increases in intracellular Ca2+ concentration ([Ca2+]i) and subsequent desensitisation were studied in CHO-K1 cells expressing human sst5 receptors (CHOsst5 cells). To study the nature of the desensitisation, interactions with uridine triphosphate (UTP) were examined. SRIF (pEC50 7.10) and UTP (pEC50) 5.14) caused concentration-dependent increases in [Ca2+]i but the SRIF maximum was about 40% of that to UTP. SRIF-, but not UTP-, induced increases in [Ca2+]i were transient and abolished by pertussis toxin. SRIF and UTP caused sustained increases in Ins(1,4,5)P3 but the SRIF maximum was about 30% of that to UTP. Removal of [Ca2+]e attenuated the SRIF-induced peak rise in [Ca2+]i but had no effect on the peak increases in Ins(1,4,5)P3. UTP-induced increases in [Ca2+]i and Ins(1,4,5)P3 were attenuated in the absence of [Ca2+]e. Following pre-exposure to SRIF (1 microM) or UTP (100 microM) for 5 min, subsequent SRIF responses were desensitised. Similar results were obtained in the absence of [Ca2+]e. Pre-exposure to SRIF had no effect on subsequent responses to UTP but in the absence of [Ca2+]e, responses to UTP were attenuated. The results suggest that SRIF but not UTP-induced increases in [Ca2+]i in CHOsst5 cells are mediated by pertussis toxin sensitive G proteins and are caused by an entry of extracellular Ca2+ and release from an Ins(1,4,5)P3 sensitive Ca2+ store. Homologous or heterologous desensitisation of agonist-induced increases in [Ca2+]i could be demonstrated in the presence or absence of extracellular Ca2+ respectively, and the latter appeared to involve depletion of a common intracellular Ca2+ store.     

Discrimination between UTP- and P2-purinoceptor-mediated depolarization of rat superior cervical ganglia by 4,4'-diisothiocyanatostilbene-2,2'- disulphonate (DIDS) and uniblue A

1. Using a grease-gap recording technique we have investigated the effects of some antagonists of P2-purinoceptors on the depolarization of the rat isolated superior cervical ganglion evoked by 100 microM alpha, beta-methylene-adenosine 5'-triphosphate (alpha,beta-MeATP) or uridine 5'-triphosphate (UTP). The effects of the putative P2Z-purinoceptor antagonist, coomassie brilliant blue G, putative P2X-purinoceptor antagonist, 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS) and uniblue A (an analogue of the P2Y- and P2X-purinoceptor antagonist reactive blue 2) were investigated. 2. At the highest concentration examined uniblue A (300 microM) depressed alpha,beta-MeATP-induced depolarization and at 100 and 300 microM enhanced UTP-evoked depolarizations. Coomassie brilliant blue G (1 and 10 microM) did not affect depolarizations evoked by alpha,beta-MeATP or UTP. Depolarizations evoked by potassium (5 mM) or muscarine (100 nM) were unaltered by either coomassie brilliant blue G or uniblue A. Uniblue A (100 and 300 microM) produced a concentration-dependent depression of hyperpolarizations evoked by adenosine (100 microM) whereas coomassie brilliant blue G at up to 10 microM, did not alter adenosine-induced hyperpolarizations. 3. DIDS (30 and 100 microM) did not alter adenosine-evoked hyperpolarizations, or depolarizations evoked by potassium or UTP. DIDS at 100 microM did not alter depolarizations evoked by muscarine. In contrast DIDS produced a concentration-dependent depression of alpha,beta-MeATP-evoked depolarizations. 4. These results are consistent with the proposal that uniblue A and DIDS but not coomassie brilliant blue G are antagonists of P2-purinoceptors and that uniblue A is also an antagonist at P1-purinoceptors present on the rat superior cervical ganglion. 5. The ability of uniblue A and DIDS to distinguish between depolarizations evoked by UTP and alpha,beta-MeATP provides further justification for the proposal that these nucleotides activate separate receptors present on the rat superior cervical ganglion, i.e. pyrimidinoceptors and P2-purinoceptors respectively.     

Purine- and pyrimidine-stimulated phosphoinositide breakdown and intracellular calcium mobilisation in astrocytes

Phosphoinositide breakdown in cultured cortical astrocytes was assessed by measuring the accumulation of [3H]inositol phosphates (IP's) following incubations with various purines and pyrimidines. Dose-response relationships gave the following order of potency: 2-methylthioadenosine triphosphate (2-MeSATP) > uridine 5'-triphosphate (UTP) > ATP = ADP > inosine 5' triphosphate (ITP). However, 2-MeSATP and UTP were only half as effective as either ATP or ADP in stimulating [3H]IP production. Astrocytes were also challenged with combined additions of maximally effective concentrations of agonists. Responses to ADP plus UTP and 2-MeSATP plus UTP were essentially additive whilst ATP plus UTP evoked a response which was only partially additive. ATP-stimulated [3H]IP accumulation was markedly reduced in the presence of 2-MeSATP suggesting that the latter may be a partial agonist at these receptors. We also examined the ability of ATP and UTP to increase intracellular Ca2+ concentrations in these cells. Greater than 90% of all cells tested responded to ATP with a release from internal Ca2+ stores but less than half of these responded similarly when challenged with UTP. Our results indicate that astrocytes possess both P2Y-purinoceptors and a population of receptors which are also coupled to phosphoinositide metabolism and intracellular Ca2+ mobilisation but recognise ATP and the pyrimidine nucleotide UTP.     

Differential heterologous and homologous desensitization of two receptors for ATP (P2y purinoceptors and nucleotide receptors) coexisting on endothelial cells

Bovine aortic endothelial cells in culture contain two coexisting phosphoinositidase C-linked receptors for ATP, the P2y-purinoceptors [for which 2-methylthio-ATP (2MeSATP) is a selective agonist] and the nucleotide (or P2u) receptors (for which UTP is a selective agonist). Here we have investigated the occurrence of homologous and heterologous desensitization of these two receptors and the involvement of protein kinase C-dependent mechanisms. Measuring total [3H]inositol (poly)phosphate accumulation in the presence of lithium, we showed that with long (15-min) stimulations with UTP or 2MeSATP desensitization occurred to a maximum of 40% within several minutes of preexposure to either agonist, i.e., with this procedure there is no difference between the heterologous and the homologous experimental design. In the remainder of the experiments reported we measured inositol-1,4,5-trisphosphate mass levels, using a protocol of 5-min preincubation, 2-min wash, and 5-sec stimulation. We found that preincubation with either agonist led to desensitization of the response to the same agonist of about 40%. However, whereas preincubation with 2MeSATP did not affect the subsequent response to UTP, preincubation with UTP did attenuate the 2MeSATP response. These results demonstrate that homologous desensitization occurs with both P2Y and nucleotide receptors but that heterologous desensitization follows only from activation of the nucleotide receptors. Preincubation with the protein kinase C inhibitor Ro 31-8220 enhanced the subsequent inositol-1,4,5-trisphosphate response to 2MeSATP but did not affect the desensitization of this response by preincubation with the same agonist. However, whereas the response to UTP was not enhanced by preincubation with the protein kinase C inhibitor, the desensitization caused by preincubation with UTP was partially inhibited by Ro 31-8220. These results show that multiple desensitizing events occur during the first few minutes of receptor activation and that these events are different for each of the receptors for ATP.     

Ca2+ signaling induced by sphingosylphosphorylcholine and sphingosine 1-phosphate via distinct mechanisms in rat glomerular mesangial cells

BACKGROUND: To elucidate the molecular mechanism underlying sphingosine 1-phosphate (S1P) and sphingosylphosphorylcholine (SPC) mediated signaling, we compared their effects with those of adenosine triphosphate (ATP) and angiotensin II (Ang II) on the cytosolic free Ca2+ concentration ([Ca2+]i), inositol 1,4, 5-trisphosphate (IP3) generation and arachidonic acid release in rat glomerular mesangial cells. METHODS: The fluorescent Ca2+ indicator, Fura-2, was used to measure the [Ca2+]i changes in cultured rat glomerular mesangial cells either in suspension or attached to the coverslips. RESULTS: SPC 5 microM, S1P 5 microM, ATP 100 microM and Ang II 90 nM all induced increases in the [Ca2+]i, and the effect showed marked homologous desensitization, while heterologous desensitization was less. After the initial exposure of the cells to SPC, the increase in [Ca2+]i induced by subsequent addition of ATP or Ang II was only reduced by about 14.3% and 4.8%, respectively. After the initial exposure to S1P, a greater reduction was seen (42. 1% and 47.7%, respectively). Both arachidonic acid release and IP3 generation were activated by all four agonists with an identical rank order of effectiveness of SPC > S1P > ATP = Ang II; both were pertussis toxin-sensitive and cholera toxin-resistant. The arachidonic acid release induced by all four agonists showed identical susceptibility to removal of extracellular Ca2+, whereas IP3 generation displayed differential extracellular Ca2+ dependence. Only SPC-induced IP3 generation was highly sensitive to extracellular Ca2+ level, and this Ca2+ dependence was abolished after pretreatment of cells with arachidonyl trifluoromethyl ketone (AACOCF3), a phospholipase A2 inhibitor. Furthermore, the Mn2+ influx was markedly greater in SPC-stimulated cells than in either control or other agonist-stimulated cells, and was decreased by prior exposure of cells to AACOCF3. After phospholipase A2 was inhibited or in the absence of extracellular Ca2+, SPC displayed identical effectiveness as S1P on desensitizing the action of ATP or Ang II on the increase in [Ca2+]i. Conclusions. Our results indicate that all four agents primarily activate phospholipase C through their receptor occupancies, but that SPC alone also induces further significant Mn2+ influx and IP3 generation attributable to its primary stimulatory effect on arachidonic acid release. Thus, the heterologous desensitization to ATP or Ang II induced by SPC was less profound than that induced by S1P, since SPC induced a Ca2+ influx.