P2U Purinoceptors Research Articles

183 MITOGEN-ACTIVATED PROTEIN KINASES ARE ACTIVATED BY ADENOSINE TRIPHOSPHATE IN RAT OVARIAN SURFACE EPITHELIAL CELLS

Extracellular ATP has been suggested to play a role in cellular proliferation and intracellular calcium concentrations (Ca2+) in ovarian cells. To investigate the role of ATP in rat ovarian folliculogenesis or steroidogenesis, we examined the expression of the P2U purinoceptor (P2U-R) and the effect of ATP on growth stimulation in rat ovarian surface epithelial (OSE) cells. Rat OSE cells were isolated and cultured in DMEM media with 10% fetal bovine serum. Our results indicated that P2U-R mRNA was expressed and that ATP exerted a growth-stimulatory effect in rat OSE cells. To investigate the mechanism of the growth-stimulatory effect, we examined the activation of mitogen-activated protein kinases (MAPK) by ATP. Treatment with ATP resulted in MAPK activation in these cells, whereas the stimulatory effect of ATP in cellular proliferation and MAPK activation was completely abolished in the presence of PD98059 [a MAPK/ERK kinase (MEK) inhibitor] and staurosporin [a protein kinase C (PKC) inhibitor], suggesting that the growth-stimulatory effect of ATP may be mediated via PKC-dependent MAPK activation in rat OSE cells. In a time-dependent study, ATP significantly increased MAPK activity at 5 to 20 min, and the activated MAPK in these cells declined to the control level after 20 min. Similarly, treatment with ATP significantly induced MAPK activation after 5 min and sustained it for 60 min in these OSE cells. In addition, treatment with ATP resulted in substantial phosphorylation of Elk-1, confirming that ATP action is mediated by activation of MAPK. In conclusion, we demonstrated that P2U-R was expressed and that ATP induced growth stimulation in rat OSE cells. Furthermore, treatment with ATP resulted in activation of the MAPK cascade and phosphorylation of Elk-1 in these cells. Taken together, these results suggest that the MAPK cascade may be involved in growth stimulation in response to ATP in rat OSE cells. This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST; No. 2010-0003093).

P2U purinoceptors: cDNA cloning, signal transduction mechanisms and structure-function analysis.

The cloning of a P2U purinoceptor cDNA has made it possible to use molecular biological approaches to investigate P2U purinoceptor function. Expression of recombinant P2U purinoceptors in mammalian cells lacking endogenous P2U purinoceptors has enabled us to characterize the receptor protein and its downstream effectors, and has allowed a partial analysis of the role of certain amino acid residues in ligand binding. These approaches have placed the pharmacological classification of the P2U purinoceptor on a firm molecular footing and have generated model systems that can be used to investigate receptor-ligand binding, regulation and signal transduction.

Paracrine stimulation of vascular smooth muscle proliferation by diadenosine polyphosphates released from proximal tubule epithelial cells

The purinergic receptor system plays an important role in the regulation of both vascular and tubular functions within the kidney; however, the release of purinergic agonists other than ATP by renal tissue is not known. In this investigation, we determine if kidney tissue is a source of diadenosine polyphosphates, which have high affinity for the P(2X) and P(2Y) receptors. Both diadenosine pentaphosphate and hexaphosphate were identified by matrix-assisted laser desorption ionization-mass spectrometry in extracts purified from both whole porcine kidney and from cloned cells of the LLC-PK1 cell line. Both polyphosphates in nanomolar concentrations were found to significantly stimulate the proliferation of vascular smooth muscle cells derived from rat thoracic aortas. The purinergic-receptor antagonist, suramin, did not significantly affect the growth-stimulatory properties of the polyphosphates. The growth stimulation of vascular smooth muscle cells by platelet-derived growth factor was potentiated by both diadenosine polyphosphates. We conclude that diadenosine polyphosphates are endogenous purinergic agonists of the kidney that have physiologic and pathophysiologic relevance. These epithelial cell metabolic products have vasoregulatory properties while linking the energy supply and tubular function.

Adenosine triphosphate activates mitogen-activated protein kinase in pre-neoplastic and neoplastic ovarian surface epithelial cells.

To investigate the role of ATP in ovarian tumorigenesis, the present study examined the expression of the P2U purinoceptor (P2U-R) and effect of ATP on growth stimulation in pre-neoplastic and neoplastic ovarian surface epithelial (OSE) cells. The immortalized OSE (IOSE) cell lines, including IOSE-29 (pre-neoplastic), IOSE-29EC (neoplastic), and OVCAR-3 (ovarian adenocarcinoma cell line) were used. Our results indicated that P2U-R mRNA was expressed and that ATP exerted a growth-stimulatory effect in IOSE-29, IOSE-29EC, and OVCAR-3. To investigate the mechanism of the growth-stimulatory effect, the activation of mitogen-activated protein kinases (MAPKs) by ATP was examined. Treatment with ATP resulted in MAPK activation in IOSE-29 and IOSE-29EC cells, whereas the stimulatory effect of ATP in cellular proliferation and MAPK activation was completely abolished in the presence of PD98059 (an MAPK/ERK kinase inhibitor) and staurosporin (a protein kinase C inhibitor), suggesting that the growth stimulatory effect of ATP is mediated via protein kinase C-dependent MAPK activation in pre-neoplastic and neoplastic OSE cells. In a time-dependent study, ATP significantly increased MAPK activity at 5-20 min in IOSE-29 cells. Activated MAPK declined to control levels after 20 min in these cells. Treatment with ATP significantly induced MAPK activation after 5 min and was sustained for 60 min in IOSE-29EC cells. In addition, treatment with ATP resulted in substantial phosphorylation of Elk-1, the Ets family transcriptional factor, confirming that ATP action is mediated by activation of MAPK. In conclusion, we have demonstrated that P2U-R was expressed and that ATP induced growth stimulation in IOSE and OVCAR-3 cells. Furthermore, treatment with ATP resulted in the activation of an MAPK cascade and phosphorylation of Elk-1 in IOSE-29 and IOSE-29EC cells. These results suggest that the MAPK cascade may be involved in growth stimulation in response to ATP in pre-neoplastic and neoplastic OSE cells.

Antigonadotropic action of adenosine triphosphate in human granulosa-luteal cells: involvement of protein kinase Calpha.

The presence of P2U purinoceptor in human granulosa-luteal cells (hGLCs) indicates a potential role of ATP in regulating ovarian function. In this study an inhibitory effect of ATP on hCG-induced cAMP production was observed. Extracellular ATP has been shown to activate protein kinase C (PKC) after binding to a purinoceptor. To understand the role of PKC in mediating ATP action, hCG-stimulated cAMP level was examined in the presence of the PKC activator, 1 micromol/L phorbol 12-myristate 13-acetate (PMA), or the PKC inhibitor, 1 micromol/L staurosporin or 1 micromol/L bisindolylmaleimide I. PMA, like 10 micromol/L ATP, significantly reduced hCG-evoked cAMP production. In addition, the inhibitory effect of ATP was reversed by staurosporin and bisindolylmaleimide I. To further investigate the involvement of PKC isoforms in mediating the inhibitory effect of ATP, the presence of PKC isoforms in cultured hGLCs was examined by Western blot using monoclonal antibodies against specific isoforms. Translocation of PKC isoforms from cytosolic fraction to membrane fraction was studied to identify the active PKC isozymes subsequent to ATP treatment. The change in PKC isoform in PKC-depleted cells (achieved by exposure to PMA for 18 h) was also examined. Our results demonstrated the presence of PKCalpha, -delta, -iota, and -lambda isoforms in hGLCs and the translocation of PKCalpha subsequent to ATP treatment. In PKC-depleted cells the PKCalpha level was reduced, and no significant effect of ATP on hCG-stimulated cAMP production was observed. To our knowledge, this is the first demonstration of PKC isoforms in hGLCs and the involvement of activated PKC in mediating the antigonadotropic effect of extracellular ATP. Taken together, these results further support a role of this neurotransmitter in regulating human ovarian function.

Expression and regulation of P2U-purinergic receptor in human granulosa-luteal cells.

The P2U purinoceptor (P2UR) has been identified pharmacologically in the ovary. However, the expression and regulation of the P2UR messenger RNA (mRNA) in human ovarian cells are still poorly characterized. The present study was designed to examine the expression and regulation of the P2UR in human granulosa-luteal cells (hGLCs) by RT-PCR and Northern blot analysis. A PCR product corresponding to the expected 599-bp P2UR complementary DNA was obtained from hGLCs. Molecular cloning and sequencing of the PCR product revealed an identical sequence to the reported P2UR complementary DNA. Two mRNA transcripts of 2.0 kb and 4.6 kb were identified in hGLCs using Northern blot analysis. The expression of the P2UR mRNA was down-regulated by human CG in a dose- and time-dependent manner. Treatment with 8-bromo-cAMP and forskolin also attenuated P2UR mRNA levels. Calcium signaling following the activation of the P2UR in single hGLCs was studied using microspectrofluorimetry. It revealed that, like ATP, uridine triphosphate (UTP) also induced cytosolic calcium mobilization in a dose-dependent manner. These results demonstrate for the first time that the P2UR mRNA is expressed in hGLCs and that P2UR mRNA is regulated by human CG, cAMP, and forskolin. The P2UR expressed in hGLCs functional because activation of the P2UR by ATP or UTP resulted in rapid and transient mobilization of cytosolic calcium at the single cell level. These findings further support a potential role of this neurotransmitter receptor in the human ovary.

Neurotransmitter ATP and cytokine release

Microglia, the resident macrophages in the central nervous system (CNS), are rapidly activated upon trauma or ischemic injury, releasing cytokines and undertaking tissue repair. Recent studies have indicated that CNS immune cells express ionotropic P2X and metabotropic P2Y purinoceptors and undergo functional changes in response to extracellular ATP. Non-stimulated cultured rat retinal microglia expressed metabotropic P2U(P2Y2, P2Y4) and ionotropic P2Z(P2X7) purinoceptors equally, whereas in LPS-stimulated microglia, P2Z and its CA2+ response became dominant. Upon hypoxia (1% oxygen) activation, the P2U response became dominant, and proliferation was induced possibly via intracellular Ca2+ mobilization and/or capacitative Ca2+ entry. TNF-alpha and IL-1 beta were released in both LPS- and hypoxia-activated states, enhanced by the P2Z agonist BzATP and suppressed by the antagonist oATP, indicating P2Z involvement in their release. P2Z activation was simultaneously anti-mitotic and induced apoptosis of microglia. Release of cytokines may be induced via Ca2+ influx and activation of NFAT, NF-kappa B or p44/42 and p38 MAP kinases, switching off the mitotic signal transduction pathway and triggering the apoptotic cascade at the same time.

ATP-induced Ca2+ response mediated by P2U and P2Y purinoceptors in human macrophages: signalling from dying cells to macrophages.

The activation of macrophages by various stimuli leading to chemotactic migration and phagocytosis is known to be mediated by an increase in intracellular Ca2+ concentration ([Ca2+]i). We measured changes in [Ca2+]i using a Ca2+ imaging method in individual human macrophages differentiated from freshly prepared peripheral blood monocytes during culture of 1-2 days. A transient rise in [Ca2+]i (duration 3-4 min) occurred in 10-15 macrophages in the vicinity of a single tumour cell that was attacked and permeabilized by a natural killer cell in a dish. Similar Ca2+ transients were produced in 90% of macrophages by application of supernatant obtained after inducing the lysis of tumour cells with hypo-osmotic treatment. Ca2+ transients were also evoked by ATP in a dose-dependent manner between 0.1 and 100 microm. The ATP-induced [Ca2+]i rise was reduced to less than one-quarter in Ca2+-free medium, indicating that it is mainly due to Ca2+ entry and partly due to intracellular Ca2+ release. UTP (P2U purinoceptor agonist) was more potent than ATP or 2-chloro-ATP (P2Y agonist). Oxidized ATP (P2Z antagonist) had no inhibitory effect. Both cell lysate- and ATP-induced Ca2+ responses were inhibited by Reactive Blue 2 (P2Y and P2U antagonist) to the same extent, but were not affected by PPADS (P2X antagonist). Sequential stimuli by cell lysate and ATP underwent long-lasting desensitization in the Ca2+ response to the second stimulation. The present study supports the view that macrophages respond to signal messengers discharged from damaged or dying cells to be ingested, and ATP is at least one of the messengers and causes a [Ca2+]i rise via P2U and P2Y receptors.

Effects of P2 purinoceptor agonists on membrane potential and intracellular Ca2+ of human cardiac endothelial cells.

Vasoactive agonists like adenosine-5'-triphosphate (ATP) increase intracellular Ca2+ ([Ca2+]i) in vascular endothelial cells with an initial peak due to inositol 1,4,5-triphosphate-mediated Ca2+ release from intracellular stores followed by a sustained plateau that is dependent on the presence of extracellular Ca2+, thus leading to an increased synthesis and release of prostacyclin and nitric oxide. We studied the effects of nucleotides on membrane potential and [Ca2+]i in confluent human microvascular cardiac endothelial cells obtained from patients with dilated cardiomyopathy. The whole-cell configuration of the patch-clamp technique and a confocal laser scanning microscope employing fluo-3 as a Ca2+ indicator were used. Both uridine-5'-triphosphate (UTP) and 2-methylthioadenosine-5'-triphosphate (2MeSATP) induced depolarizations in human microvascular cardiac endothelial cells and increased [Ca2+]i with a rank order of potency 2MeSATP>ATP=UTP (EC50 values (in microM) were 0.084 2MeSATP, 0.67 ATP and 1.1 UTP). This suggests that both P2u and P2y purinoceptors are present on human microvascular cardiac endothelial cells. Maximal [Ca2+]i responses of confluent human microvascular cardiac endothelial cell monolayers to UTP were lower when compared to 2MeSATP. Nucleotide-induced increases in [Ca2+]i consisted of a transient peak, which was also observed in the absence of extracellular Ca2+, and a sustained [Ca2+]i plateau. This plateau, which was not observed in all monolayers studied, was not markedly influenced by increasing extracellular [K+]. Previous incubation with thapsigargin abolished ATP-induced increases of [Ca2+]i. It is concluded that human microvascular cardiac endothelial cells express both P2y and P2u purinoceptors. P2 purinoceptor agonists release Ca2+ from intracellular thapsigargin-sensitive stores and stimulate capacitative Ca2+ influx pathways. K+ efflux through Ca2+-dependent K+ (K(Ca)) channels does not play a major role in the regulation of nucleotide-induced Ca2+ influx in human microvascular cardiac endothelial cells, which might be related to an impaired function of the cells.

Intracellular calcium oscillations induced by ATP in airway epithelial cells.

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

Erythromycin inhibits ATP-induced intracellular calcium responses in bovine tracheal epithelial cells.

Erythromycin (EM) therapy is known to decrease airway secretion in chronic inflammatory airway diseases such as diffuse panbronchiolitis. Airway secretion is regulated by intracellular Ca2+ concentration ([Ca2+]i). To elucidate the intracellular site of action of EM in airway epithelium, we examined the effect of EM on Ca2+ dynamics in cultured bovine tracheal epithelial cells using fura-2. EM per se did not cause any change in [Ca2+]i. Adenosine triphosphate (ATP; 10(-4) M) induced a biphasic [Ca2+]i increase, consisting of a transient response followed by a sustained response. Pretreatment of cells with EM had little effect on the ATP-induced transient Ca2+ response but substantially reduced the sustained response in a dose-dependent manner. Clarithromycin, another 14-membered ring macrolide, likewise showed the inhibitory effect, but ampicillin and cephasolin did not. Uridine triphosphate (UTP; 10(-4) M) induced a biphasic [Ca2+]i increase similar to ATP, and the UTP-induced sustained Ca2+ response was also inhibited by EM. In Ca2+-deficient medium (1 mM ethyleneglycol-bis-(beta-aminoethyl ether)-N, N'-tetraacetic acid [EGTA]) or in the presence of La3+, the sustained Ca2+ response disappeared, suggesting that EM may inhibit Ca2+ influx induced by P2u purinoceptor stimulation. In single-cell Ca2+ image analysis, low concentration of ATP (10(-6) M) induced Ca2+ oscillations, which were also inhibited by EM. The disappearance of [Ca2+]i oscillations after addition of EM was similar to that after addition of EGTA. These results suggest that EM may decrease Ca2+-dependent airway secretion by inhibiting agonist-stimulated Ca2+ influx.

Human 5-HT1B receptor stimulated inositol phospholipid hydrolysis in CHO cells: synergy with Gq-coupled receptors

We have previously reported that the transfected Gi/Go protein-coupled human adenosine A1 receptor (expressed at 200 fmol/mg of protein) and the endogenous 5-HT1B receptor (not detectable using radioligand binding) suppress forskolin-stimulated cyclic AMP accumulation and stimulate increases in [Ca2+]i in Chinese hamster ovary cells (CHO). In addition, co-activation of the adenosine A1 receptor (but not the 5-HT1B receptor) potentiates the hydrolysis of inositol phospholipids elicited by receptors coupled to Gq-proteins (Dickenson and Hill, 1996. Eur. J. Pharmacol. 320, 141-151). In order to establish whether this difference in ability to modulate Gq-coupled receptor responses is a consequence of low 5-HT1B receptor density, we have stably transfected CHO-KI cells with the human 5-HT1Dbeta cDNA (the human homologue of the rodent 5-HT1B receptor). We initially isolated a clonal cell line (designated CHO5-HT1B cells) displaying moderate specific [3H]5-HT binding (pKd of 8.17+/-0.07 and a Bmax of 140 fmol/mg protein). In CHO5-HT1B cells, the selective human 5-HT1B/1D receptor agonist sumatriptan produced a concentration-dependent inhibition of forskolin-stimulated cyclic AMP accumulation (pEC50=7.92+/-0.04). Sumatriptan also elicited a moderate and pertussis toxin-sensitive increase in [3H]inositol phosphate formation in CHO-5HT1B cells (pEC50=6.51+/-0.05). Finally, sumatriptan synergistically enhanced P2U purinoceptor stimulated [3H]inositol phosphate accumulation through a pertussis toxin-sensitive mechanism. These findings clearly show the significance of 5-HT1B receptor expression level in determining whether 5-HT1B receptor activation can modulate the accumulation of [3H]inositol phosphates elicited by a Gq-protein coupled receptor. The observation that 5-HT1B receptor activation can potentiate Gq-coupled receptor stimulated second messenger responses may have an important physiological role in the regulation of vascular smooth muscle contraction.

Calcium Mobilization Systems During Neurogenesis.

Neuroepithelial cells have Ca(2+) mobilization systems that are activated by acetylcholine via muscarinic receptors and by extracellular ATP via P(2U) purinoceptors. The Ca(2+) mobilization occurs during neurogenesis and diminishes in parallel with the declining of mitotic activities of the neuroepithelial cells. Capacitative Ca(2+) influx also occurs with the Ca(2+) mobilization.

Decline of the calcium response on successive stimulation of a rat brain endothelial cell P2U purinoceptor.

A microfluorimetric method using Fura-2 as calcium indicator was used to study the mechanism of desensitization of the calcium response evoked by activation of a brain endothelial cell P2U receptor. The study was mainly carried out on an immortalized rat brain endothelial cell line (RBE4), with some additional experiments on primary cultured rat brain microvascular endothelial cells. As previously described (Nobles et al. 1995), ATP (100 microM, 20 s) caused a transient increase in intracellular calcium levels ([Ca2+]i). This effect was dependent on the rate of filling of intracellular calcium stores, since a large inhibition of the ATP-mediated response was seen in the presence of cyclopiazonic acid, an inhibitor of the store Ca(2+)-ATPase. Application of repeated pulses of extracellular ATP led to a desensitization of the response, as measured by a decline in the release of intracellular calcium (Nobles et al. 1995). This desensitization was partially reversed after 300 s of incubation in agonist-free medium. Extracellular phosphorylation of the purinergic receptor appeared not to be involved in the desensitization process, since a similar rate of desensitization was obtained with the non-hydrolysable ATP analogue ATP gammaS. Oxidation of the purinergic receptor cannot account for the desensitization, since the decline of the ATP-mediated response was unchanged in the presence of 3 mM dithiothreitol. In the presence of ATP together with UTP, two equally potent activators of the P2U receptor, the desensitization was less than in the presence of only one of the agonists. The desensitization was greater when ATP was applied for longer (150 s) periods. Although these results do not exclude the participation of post-receptor events in the desensitization process, they suggest that desensitization is governed at least in part by agonist-receptor interaction.

Overexpression of P2Y2 purinoceptor in intimal lesions of the rat aorta.

Extracellular nucleotides, particularly ATP, are involved in the modulation of arterial vasomotricity via P2 purinoceptors present on smooth muscle and endothelial cells. These nucleotides could also be implicated in the smooth muscle cell hyperplasia observed in intimal lesions. In this study, we tried to define the potential role of the P2Y2 (P2u) purinoceptor by studying its expression in normal and balloon-injured rat aortas. The cloning of a rat P2Y2 cDNA from a rat smooth muscle cell cDNA library made it possible to study P2Y2 expression both by Northern blot and in situ hybridization. Northern blot experiments indicated that P2Y2 mRNA was present in rat medial aortic smooth muscle and in cultured rat aortic smooth muscle cells. In situ hybridization indicated that P2Y2 mRNA was present in endothelial cells of the intima and in some smooth muscle cells scattered throughout the media of adult rat aortas, while almost all medial smooth muscle cells of rat embryo aorta expressed this receptor. In contrast with adult aortic media, the majority of neointimal smooth muscle cells found in aortic intimal lesions either 8 or 20 days after balloon injury were positive for P2Y2 mRNA. Moreover, a subpopulation of neointimal cells localized at the luminal surface could be identified by a higher P2Y2 expression than the underlying neointimal smooth muscle cells. These data showing a strong expression of the P2Y2 purinoceptor in the neointima of injured arteries suggest that extracellular nucleotides may be involved, via this receptor, in the intimal hyperplasia and/or chronic constriction observed at the lesion site, and consequently in the restenotic process.

Endothelial-mediated dilations of rat middle cerebral arteries by ATP and ADP.

The hypothesis that ATP and ADP produce dilations of rat middle cerebral arteries (MCAs) by different mechanisms was tested. Vessel diameters were measured from pressurized, perfused MCAs after application of different agonists. The luminal administration of ATP and ADP elicited concentration-dependent dilations (35% maximum). Removal of endothelium abolished the dilation to intraluminal ATP and attenuated the dilation to intraluminal ADP. The dilations to ATP were abolished with N omega-nitro-L-arginine methyl ester (L-NAME; 10 microM), a nitric oxide synthase inhibitor, at ATP concentrations of 1 microM and below. However, at concentrations of 10 microM ATP and above, L-NAME had no effect on the response. The dilations to ADP were attenuated by L-NAME to the same degree as removal of endothelium. The mechanism for dilation by ATP was identical to that of UTP, a selective P2u purinoceptor agonist. The mechanism of dilation by ADP was similar to that of 2-methylthioadenosine 5'-triphosphate, a selective P2y purinoceptor agonist. We conclude that ATP and ADP elicit dilations of rat MCA by different mechanisms. ATP and ADP likely stimulate P2u and P2y purinoceptors, respectively.

Role of purinergic receptors in chloride secretion in Caco-2 cells.

Purinergic receptors play an important role in regulating Cl- secretion in epithelial cells. To explore further the role of these receptors in the intestine, we utilized the human intestinal epithelial cell line, Caco-2, grown on permeable membrane supports and assayed for Cl- secretion by measuring the short-circuit current (Isc). Stimulation of Isc by extracellular nucleotides could be detected by day 4 and increased by day 10 postseeding. The magnitude of stimulation of Isc at 10 microM in cells at day 10 was UTP > ATP > UDP > > 2-methylthioadenosine 5'-triphosphate (2-MeS-ATP) = ADP on the apical side and UTP = 2-MeS-ATP = ATP > ADP > > UDP on the basolateral side. Cross-desensitization studies suggested that two different receptors are expressed in the apical membrane, a P2U purinoceptor and a uridine nucleotide receptor. Two different receptors are also expressed in the basolateral membrane, a P2U receptor and another that reacts with both 2-MeS-ATP and ADP. This latter receptor has an unusual pharmacological profile, with a reactivity for 2-MeS-ATP > ADP but not for ATP. Responses to purinergic receptor agonists were inhibited by pretreatment with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester, thapsigargin, or quinine. Thus we suggest that an increase in intracellular Ca2+ and subsequent opening of Ca(2+)-activated K+ channel play a role in increasing driving force for Cl- to exit across the apical membrane. The role of the cystic fibrosis transmembrane conductance regulator as a Cl- exit pathway on the apical membrane was also established.

Confocal imaging analysis of ATP-induced Ca2+ response in individual endothelial cells of the artery in situ.

The mechanisms for mobilization of intracellular free Ca2+ have been studied in various types of isolated and cultured cells, but little is known about Ca2+ mobilization in individual cells in situ. We tried to establish imaging analysis of intracellular free Ca2+ concentration ([Ca2+]i) in individual cells loaded with the acetoxymethyl ester of fluo 3 in situ, using laser scanning confocal microscopy. The method permitted us to distinguish signals from endothelial and smooth muscle cells of guinea pig artery. Addition of ATP to the artery caused a transient increase in endothelial [Ca2+]i. It was concluded that the response was induced via P2Y purinoceptors, because adenosine 5'-O-(2-thiodiphosphate), but not UTP, caused a similar response independent of extracellular Ca2+. The percentage of cells that responded to ATP (1-10 microM) and the peak amplitude of the transient increase in [Ca2+]i were dose dependently increased. Using rapid xy-scanning and line-scanning modes, we confirmed that 10 microM ATP induced Ca2+ waves, at a rate of 10-30 microns/s, after a lag time of approximately 3 s. These results show that [Ca2+]i waves within endothelial cells are physiologically induced by ATP via P2Y purinoceptor, but not P2U purinoceptor, in aortic strips in situ. The method should be of use in the study of vascular physiology and pathophysiology.

The P2U Purinoceptor Obligatorily Engages the Heterotrimeric G Protein G16 to Mobilize Intracellular Ca2+ in Human Erythroleukemia Cells

To assess the role of G16, a trimeric G protein exclusively expressed in hematopoietic cells, Galpha16 antisense RNA was stably expressed in human erythroleukemia (HEL) cells. Western blot analysis showed that in transfected cell lines, the expression of endogenous Galpha16 protein was suppressed, but the expression of Galphaq/11, Galphai2, and Galphai3 remained unaffected. Suppression of Galpha16 in transfected HEL cells did not interfere with transient elevations of intracellular free Ca2+ concentrations induced by prostaglandin E1 (PGE1), platelet-activating factor, or thrombin. In parental HEL cells, UTP and ATP mobilized Ca2+ from intracellular stores with half-maximum effective concentrations of 3. 6 +/- 0.7 and 4.7 +/- 1.6 microM, respectively, apparently by stimulating P2U purinoceptors. By contrast, Ca2+ mobilization by UTP or ATP was completely abrogated in Galpha16-suppressed cells, indicating specific coupling of G16 to P2U purinoceptors. Pertussis toxin inhibited the effect of UTP in parental HEL cells by 57.6 +/- 4.9%. These data indicate that signaling by the P2U purinoceptor obligatorily requires G16 but may be modulated further by activation of Gi. Priming of HEL cells with UTP or ATP prior to stimulation with PGE1 markedly enhanced the PGE1-induced intracellular Ca2+ release. This indirect, potentiating effect of UTP and ATP was not impaired in Galpha16-suppressed cells but was inhibited by pertussis toxin, indicating that functional P2U purinoceptors are present on these cells and that the potentiating effect primarily depends on Gi. The data demonstrate (i) that Galpha16 antisense RNA selectively inhibits endogenous Galpha16 protein expression in HEL cells; (ii) that stimulation of endogenous P2U (P2Y2) purinoceptors leads to the mobilization of intracellular Ca2+ by a mechanism that strictly depends on Galpha16; and (iii) that P2U purinoceptors in HEL cells can communicate with two distinct signaling pathways diverging at the G protein level.

Two distinct P2 purinergic receptors, P2Y and P2U, are coupled to phospholipase C in mouse pineal gland tumor cells.

We found that extracellular ATP can increase the intracellular Ca2+ concentration ([Ca2+]i) in mouse pineal gland tumor (PGT-beta) cells. Studies of the [Ca2+]i rise using nucleotides and ATP analogues established the following potency order: ATP, adenosine 5'-O-(3-thiotriphosphate) > or = UTP > 2-chloro-ATP > 3'-O-(4-benzoyl)benzoyl ATP, GTP > or = 2-methylthio ATP, adenosine 5'-O-(2-thiodiphosphate) (ADP beta S) > CTP. AMP, adenosine, alpha,beta-methyleneadenosine 5'-triphosphate, beta,gamma-methyleneadenosine 5'-triphosphate, and UMP had little or no effect on the [Ca2+]i rise. Raising the extracellular Mg2+ concentration to 10 mM decreases the ATP- and UTP-induced [Ca2+]i rise, because the responses depend on the ATP4- and UTP4- concentrations, respectively. The P2U purinoceptor-selective agonist UTP and the P2Y purinoceptor-selective agonist ADP beta S induce inositol 1,4,5-trisphosphate generation in a concentration-dependent manner with maximal effective concentrations of approximately 100 microM. In sequential stimulation, UTP and ADP beta S do not interfere with each other in raising the [Ca2+]i. Costimulation with UTP and ADP beta S results in additive inositol 1,4,5-trisphosphate generation to a similar extent as is achieved with ATP alone. Pretreatment with pertussis toxin inhibits the action of UTP and ATP by maximally 45-55%, whereas it has no effect on the ADP beta S response. Treatment with 1 microM phorbol 12-myristate 13-acetate inhibits the ADP beta S-induced [Ca2+]i rise more effectively than the ATP- and UTP-induced responses. These results suggest that P2U and P2Y purinoceptors coexist on PGT-beta cells and that both receptors are linked to phospholipase C.