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

1. The alpha 1-adrenoceptor subtypes mediating contractions of the smooth muscle in human prostatic urethra and branches of internal iliac artery were characterized in isometric contraction experiments. 2. Phenylephrine produced concentration-dependent contractions in both the urethra and artery. These responses were competitively inhibited by prazosin, WB4101 and 5-methyl-urapidil, and the slopes of Schild plots for all these antagonists were close to unity. 3. The pA2 values for prazosin were not significantly different between the urethra (9.42 +/- 0.11; mean +/- s.d.) and artery (9.50 +/- 0.27), while the pA2 values for WB4101 and 5-methyl-urapidil in the human prostatic urethra (8.94 +/- 0.19 and 8.42 +/- 0.14, respectively) were significantly greater than in the branches of human internal iliac artery (7.94 +/- 0.21 and 7.43 +/- 0.22, respectively; P < 0.01). 4. Pretreatment with chlorethylclonidine (CEC) at concentrations ranging from 0.1 microM to 100 microM attenuated the maximum contraction to phenylephrine in a concentration-dependent manner in both the urethra and artery. However, the urethra was significantly less affected by CEC than the artery. The pD'2 values (negative logarithm of the molar concentration of antagonist which reduced the maximum contraction to one half) in the urethra and artery were 4.35 +/- 0.27 and 5.20 +/- 0.37, respectively (P < 0.01). 5. The present results indicate that there are distinct populations of alpha 1-adrenoceptor subtypes in the human prostatic urethra and branches of the internal iliac artery.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for a nucleotide receptor on adrenal medullary endothelial cells linked to phospholipase C and phospholipase D.

1. We have investigated whether the 'atypical' P2-purinoceptor previously described on adrenal microvasculature endothelial cells is a nucleotide receptor (responds to pyrimidines and purines) and is linked to phospholipase D as well as phospholipase C. 2. Cultured bovine adrenal medullary endothelial (BAME) cells responded to the pyrimidine UTP, as well as the purines. The total [3H]-inositol phosphate responses were with a rank order of UTP > ATP- = adenosine 5'-O-(3-thio-triphosphate) (ATP gamma S) >> 2MeSATP. The selective P2x agonist beta, gamma-methylene ATP was inactive. 3. Construction of dose-response curves to ATP, ATP gamma S and UTP in the presence and absence of additional agonists showed that responses to ATP gamma S and UTP were not additive, nor were those to UTP and ATP. This suggests that purines and pyrmidines acted via a common nucleotide receptor. 4. 32P-labelled BAME cells, in the presence of butanol, produced [32P]-phosphatidylbutanol (PBut) when stimulated with ATP gamma S or the protein kinase C activator, tetradecanoyl phorbol acetate (TPA). 5. Cells labelled with [3H]-palmitate and stimulated in the presence of butanol generated [3H]-PBut with the same order of agonist potencies seen for inositol phosphate responses. 6. The protein kinase C inhibitor, Ro 31-8220, abolished TPA and agonist stimulation of [3H]-PBut production. 7. These observations, and our related studies on bovine aortic endothelial cells, provide the first demonstration of a phospholipase C linked nucleotide receptor on vascular endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of brain capillary endothelial cells by P2Y receptors coupled to Ca2+, phospholipase C and mitogen-activated protein kinase

1. The blood-brain barrier is formed by capillary endothelial cells and is regulated by cell-surface receptors, such as the G protein-coupled P2Y receptors for nucleotides. Here we investigated some of the characteristics of control of brain endothelial cells by these receptors, characterizing the phospholipase C and Ca²⁺ response and investigating the possible involvement of mitogen-activated protein kinases (MAPK).
2. Using an unpassaged primary culture of rat brain capillary endothelial cells we showed that ATP, UTP and 2-methylthio ATP (2MeSATP) give similar and substantial increases in cytosolic Ca²⁺, with a rapid rise to peak followed by a slower decline towards basal or to a sustained plateau. Removal of extracellular Ca²⁺ had little effect on the peak Ca²⁺-response, but resulted in a more rapid decline to basal. There was no response to α,β-MethylATP (α,βMeATP) in these unpassaged cells, but a response to this P2X agonist was seen after a single passage.
3. ATP (log EC₅₀ −5.1±0.2) also caused an increase in the total [³H]-inositol (poly)phosphates ([³H]-InsP_x) in the presence of lithium with a rank order of agonist potency of ATP=UTP=UDP>ADP, with 2MeSATP and α,βMeATP giving no detectable response.
4. Stimulating the cells with ATP or UTP gave a rapid rise in the level of inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃), with a peak at 10 s followed by a decline to a sustained plateau phase. 2MeSATP gave no detectable increase in the level of Ins(1,4,5)P₃.
5. None of the nucleotides tested affected basal cyclic AMP, while ATP and ATPγS, but not 2MeSATP, stimulated cyclic AMP levels in the presence of 5 μM forskolin.
6. Both UTP and ATP stimulated tyrosine phosphorylation of p42 and p44 mitogen-activated protein kinase (MAPK), while 2MeSATP gave a smaller increase in this index of MAPK activation. By use of a peptide kinase assay, UTP gave a substantial increase in MAPK activity with a concentration-dependency consistent with activation at P2Y₂ receptors. 2MeSATP gave a much smaller response with a lower potency than UTP.
7. These results are consistent with brain endothelial regulation by P2Y₂ receptors coupled to phospholipase C, Ca²⁺ and MAPK; and by P2Y₁-like (2MeSATP-sensitive) receptors which are linked to Ca²⁺ mobilization by a mechanism apparently independent of agonist stimulated Ins (1,4,5)P₃ levels. A further response to ATP, acting at an undefined receptor, caused an increase in cyclic AMP levels in the presence of forskolin. The differential MAPK coupling of these receptors suggests that they exert fundamentally distinct influences over brain endothelial function.

     

Neuronal "nucleotide" receptor linked to phospholipase C and phospholipase D? Stimulation of PC12 cells by ATP analogues and UTP.

We have investigated the characteristics of the receptor for ATP on neuronal cells and the involvement of phospholipase C and phospholipase D in the effector mechanisms, using PC12 rat phaeochromocytoma cells in culture. We show that the cells respond, with generation of total inositol phosphates, to ATP and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) but not to 2-methylthioadenosine5'-triphosphate (2MeSATP), beta,gamma-methylene ATP, or adenosine 5'-O-(2-thiodiphosphate) (ADP beta S). The largest response to ATP gamma S was mainly independent of extracellular calcium, had an EC50 of 7.93 +/- 0.76 microM, and was competitively inhibited by the nonspecific antagonist suramin. The pyrimidine nucleotide UTP also elicited a response in these cells. Measurement of [3H]inositol triphosphate showed a rapid rise to maximum (10-15 sec) in response to both ATP gamma S and UTP but no response to 2MeSATP. Cells prelabeled with 32Pi and stimulated in the presence of 50 mM butanol responded to ATP gamma S, ATP, and UTP with enhanced formation of [32P]phosphatidylbutanol as well as [32P]phosphatidic acid, indicating that agonist-stimulated phosphatidic acid occurs by both phospholipase D and phospholipase C activity. The stimulation of phospholipase D was inhibited by the presence of a protein kinase C inhibitor, Ro 31-8220. The dose-response curve for the stimulation by ATP gamma S of phospholipase C was shifted to the right by the presence of UTP, indicating that both compounds act on the same receptors. The data provide the first evidence for the existence of a nucleotide receptor on neuronal cells (insensitive to both purines and pyrimidines) and show that this receptor is linked to both phospholipase C and phospholipase D.     

Protein kinase C isoforms in bovine aortic endothelial cells: role in regulation of P2Y- and P2U-purinoceptor-stimulated prostacyclin release.

1. Enhanced synthesis of prostacyclin (PGI2) and inositol polyphosphates in bovine aortic endothelial cells in response to ATP and ADP is mediated by co-existing P2Y- and P2U-purinoceptors. Here we examine the regulation of these responses by isoforms of protein kinase C (PKC). 2. Immunoblots with antisera specific for 8 different PKC isoforms revealed the presence of alpha, epsilon and zeta, while no immunoreactivity was found for beta, gamma, delta, eta and theta isoforms. PKC-alpha was largely cytosolic in unstimulated cells and almost all translocated to the membrane (Triton X-100 soluble) after a 1 min treatment with the PKC activating phorbol myristate acetate (PMA); PKC-epsilon was always in a Triton X-100 insoluble membrane fraction, while PKC-zeta was found in both soluble and membrane bound (Triton X-100 soluble) forms in the unstimulated cells and was unaffected by PMA. 3. Treatment with PMA for 6 h led to a 90% downregulation of PKC-alpha, while the immunoreactivity to the epsilon and zeta isoforms remained largely unchanged. 4. After either 10 min or 6 h exposure to PMA the PGI2 response to activation of both receptors was enhanced, while the inositol 1,4,5-trisphosphate response to P2Y-purinoceptor activation was substantially attenuated and the P2U-purinoceptor response was unchanged. Thus the PGI2 response to PMA under conditions when 90% of the PKC-alpha was lost resembles that seen on acute stimulation of PKC by PMA, and the PGI2 response does not correlate with phospholipase C response. 5. Inhibition of PKC with the isoform non-selective inhibitors, Ro 31-8220 and Go 6850 abolished the PGI2 response to both P2U- and P2Y-purinoceptor stimulation. However, Go 6976, which preferentially inhibits Ca2+ sensitive isoforms (such as PKC-alpha) and not Ca2+ insensitive isoforms (such as PKC-epsilon), had no effect on the PGI2 response. 6. The results show that there is a requirement for PKC in the stimulation of PGI2 production by endothelial P2Y- and P2U-purinoceptors. Both downregulation and inhibition studies show that PKC-alpha is not responsible for the regulation of the response to P2-purinergic stimulation, and imply that the response is mediated by PKC-epsilon (PKC-zeta is unresponsive to PMA), or an as yet uncharacterized PKC isoform.     

Evidence that a form of ATP uncomplexed with divalent cations is the ligand of P2y and nucleotide/P2u receptors on aortic endothelial cells.

1. The response of bovine aortic endothelial cells to adenosine 5'-triphosphate (ATP) is mediated by both P2y and nucleotide/P2u receptors. In order to determine which form of the nucleotide is the true ligand of these receptors, we have investigated the effects of divalent cations on ATP-, uridine 5'-triphosphate (UTP)- and 2 methylthioadenosine 5'-triphosphate (2MeSATP)-induced inositol phosphate accumulation in these cells. 2. Omisson of Mg2+ from a calcium-free incubation buffer caused a shift to the left of the ATP concentration-action curve. 3. In the presence of EDTA (1 mM), the basal level of inositol trisphosphate (InsP3) was markedly increased and the absolute maximal response to ATP was decreased; however, the response to low concentrations of ATP was enhanced. 4. When the results were plotted in terms of calculated ATP4- concentrations, the concentration-response curves obtained in the presence of 1.25 mM Mg2+ lay closer to the respective curves obtained when Mg2+ was omitted from the medium or when Mg2+ was omitted and EDTA (1 mM) was added. The curves became almost superimposable when the baseline value was subtracted. 5. A similar shift to the left of the concentrations-action curves was also observed with both UTP and 2MeSATP. 6. Our data provide evidence that a form of ATP uncomplexed with divalent cation is the preferential agonist of both the nucleotide/P2u and the P2y receptors expressed on bovine aortic endothelial cells.     

Anandamide initiates Ca(2+) signaling via CB2 receptor linked to phospholipase C in calf pulmonary endothelial cells

The endocannabinoid anandamide has been reported to affect neuronal cells, immune cells and smooth muscle cells via either CB1 or CB2 receptors. In endothelial cells, the receptors involved in activating signal transduction are still unclear, despite the fact that anandamide is produced in this cell type. The present study was designed to explore in detail the effect of this endocannabinoid on Ca2+ signaling in single cells of a calf pulmonary endothelial cell line. Anandamide initiated a transient Ca2+ elevation that was prevented by the CB2 receptor antagonist SR144528, but not by the CB1 antagonist SR141716A. These data were confirmed by molecular identification of the bovine CB2 receptor in these endothelial cells by partial sequencing. The phospholipase C inhibitor 1-[6-[[(17beta)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5dione and the inositol 1,4,5-trisphosphate receptor antagonist 2-aminoethoxydiphenylborate prevented Ca2+ signaling in response to anandamide. Using an improved cameleon probe targeted to the endoplasmic reticulum (ER), fura-2 and ratiometric-pericam, which is targeted to the mitochondria, anandamide was found to induce Ca2+ depletion of the ER accompanied by the activation of capacitative Ca2+ entry (CCE) and a transient elevation of mitochondrial Ca2+. These data demonstrate that anandamide stimulates the endothelial cells used in this study via CB2 receptor-mediated activation of phospholipase C, formation of inositol 1,4,5-trisphosphate, Ca2+ release from the ER and subsequent activation of CCE. Moreover, the cytosolic Ca2+ elevation was accompanied by a transient Ca2+ increase in the mitochondria. Thus, in addition to its actions on smooth muscle cells, anandamide also acts as a powerful stimulus for endothelial cells.     

Pharmacological characterization of nucleotide P2Y receptors on endothelial cells of the mouse aorta

Nucleotides regulate various effects including vascular tone. This study was aimed to characterize P2Y receptors on endothelial cells of the aorta of C57BL6 mice. Five adjacent segments (width 2 mm) of the thoracic aorta were mounted in organ baths to measure isometric force development. Nucleotides evoked complete (adenosine 5' triphosphate (ATP), uridine 5' triphosphate (UTP), uridine 5' diphosphate (UDP); >90%) or partial (adenosine 5' diphosphate (ADP)) relaxation of phenylephrine precontracted thoracic aortic rings of C57BL6 mice. Relaxation was abolished by removal of the endothelium and was strongly suppressed (>90%) by inhibitors of nitric oxide synthesis. The rank order of potency was: UDP approximately UTP approximately ADP>adenosine 5'-[gamma-thio] triphosphate (ATPgammaS)>ATP, with respective pD2 values of 6.31, 6.24, 6.22, 5.82 and 5.40. These results are compatible with the presence of P2Y1 (ADP>ATP), P2Y2 or P2Y4 (ATP and UTP) and P2Y6 (UDP) receptors. P2Y4 receptors were not involved, since P2Y4-deficient mice displayed unaltered responses to ATP and UTP.The purinergic receptor antagonist suramin exerted surmountable antagonism for all agonists. Its apparent pKb for ATP (4.53+/-0.07) was compatible with literature, but the pKb for UTP (5.19+/-0.03) was significantly higher. This discrepancy suggests that UTP activates supplementary non-P2Y2 receptor subtype(s). Further, pyridoxal-phosphate-6-azophenyl-2'-4'-disulphonic acid (PPADS) showed surmountable (UTP, UDP), nonsurmountable (ADP) or no antagonism (ATP). Finally, 2'-deoxy-N6-methyladenosine3',5'-bisphosphate (MRS2179) inhibited ADP-evoked relaxation only. Taken together, these results point to the presence of functional P2Y1 (ADP), P2Y2 (ATP, UTP) and P2Y6 (UDP) receptors on murine aorta endothelial cells. The identity of the receptor(s) mediating the action of UTP is not fully clear and other P2Y subtypes might be involved in UTP-evoked vasodilatation.     

Stimulation of phosphatidic acid synthesis in bovine aortic endothelial cells in response to activation of P2-purinergic receptors.

In this study we used the bovine thoracic aorta endothelial cell line AG 4762 and primary bovine aortic endothelial cells to investigate the formation of phosphatidic acid (PA) in response to activation of P2-purinergic receptors. 2-Methylthio ATP (2MeSATP) stimulated the formation of [32P]-PA in bovine aortic endothelial cells labelled with 32P(i) for 2.5 hr. A comparison of the response to other ATP analogues suggests that this was mediated via a P2Y-purinergic receptor. Using various agonists at 30 microM there was a correlation between the formation of [32P]PA and of total inositol phosphates in the presence of lithium. The 2MeSATP-stimulated accumulation of [32P]PA showed an initial high rate, followed by a more sustained slower rate. The initial response was independent of extracellular calcium while the later response was dependent on calcium influx. The protein kinase C stimulator phorbol myristate acetate (PMA) produced only a very small enhancement of [32P]PA accumulation compared to 2MeSATP. The 2MeSATP stimulation of both inositol phosphates and [32P]PA was almost eliminated by the presence of PMA. Using cells prelabelled with [3H]methylcholine 2MeSATP produced only a small non-significant enhancement of [3H]choline formation; PMA by contrast formed a much larger amount of [3H]choline. There was no evidence of a change in [3H]phosphocholine. The dissociation between phospholipase D (PLD) activation and [32P]PA accumulation and the correlation between stimulation of [32P]PA accumulation and phospholipase C (PLC) activation all suggest that, using this protocol for labelling cells, the principle route of the stimulation of formation of [32P]PA is via the activation of PLC followed by metabolism of diacylglycerol (DAG) by DAG kinase. These results show that activation of P2Y-purinergic receptors on aortic endothelial cells leads to the formation of phosphatidic acid and that both PLD and PLC pathways are likely to contribute to this response.     

The nucleotide receptors on mouse C2C12 myotubes.

1. The response of C2C12 mouse myotubes to stimulation with adenosine triphosphate (ATP) and other nucleotides was studied by measuring changes in membrane potential. 2. A transient hyperpolarization followed by a slowly declining depolarization of the cells was observed in the presence of ATP (10 microM-1 mM). 3. The hyperpolarization was not observed in the absence of external calcium, and was abolished in the presence of tetraethylammonium (20 mM) or the bee toxin, apamin (0.1 microM). The depolarization was reduced under low sodium conditions. 4. A biphasic change in membrane potential was also recorded in the presence of adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) and the pyrimidine uridine triphosphate (UTP), while the ATP derivatives and analogues, adenosine diphosphate, adenosine, alpha,beta-methylene ATP and 2-methylthio ATP and the nucleotides, guanosine triphosphate and cytidine triphosphate, did not affect the membrane potential of the myotubes. 5. The hyperpolarization elicited by ATP gamma S or UTP was also blocked by apamin and abolished under Ca(2+)-free conditions. 6. In contrast to ATP and ATP gamma S, the depolarization evoked by UTP was unaffected under low Na+ and less sensitive to the antagonistic action of suramin. 7. The ATP and UTP responses at maximal concentration were not additive after simultaneous application. ATP elicited a depolarization if applied after UTP, while UTP did not change membrane potential following the application of ATP. 8. The concentration-response curves of the effective nucleotides were shifted to the right in the presence of suramin, suggesting competitive antagonism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Down-regulation of beta receptors by desipramine in vitro involves PKC/phospholipase A2.

Chronic treatment with a number of antidepressants results in a down-regulation and/or a desensitization of rat cortical beta-adrenergic receptors (beta ARs). Although these effects generally have been attributed to elevations in intrasynaptic norepinephrine via presynaptic mechanisms, the recent demonstration of similar changes in beta ARs following in vitro incubation of cultured cells with desipramine (DMI) suggests that direct, postsynaptic mechanisms may also be involved. To study these mechanisms, we incubated rat C6 glioma cells with 10 microM DMI for 1 or 5 days. DMI produced a significant reduction in beta AR density following chronic (but not acute) treatment (BMAX control = 1325 +/- 78 fmol/mg; DMI = 1179 +/- 96; p less than .05). Interestingly, the beta AR down-regulation was accompanied by an increase in KL/KH ratio (ratio of dissociation constants for the low- and high-affinity states of the receptor), suggesting that these drugs may stabilize the high-affinity complex. DMI treatment attenuated the cyclic adenosine monophosphate (cAMP) response to 1 microM isoproterenol (control = 540 +/- 82 pmol/mg/15 min; DMI = 335 +/- 64; p less than .05), but not to agents acting distal to the receptor (cholera toxin or forskolin). Coincubation of C6 cells with either the phospholipase A2 (PLA2) inhibitor mepacrine or the protein kinase C (PKC) inhibitor H7, during chronic treatment with DMI, blocked the down-regulation of beta ARs. Incubation of C6 cells with phorbol esters (PKC activators) also down-regulated beta ARs, effects that were nonadditive with those of DMI. Incubation with H7 alone resulted in an up-regulation of beta ARs, consistent with a tonic regulatory effect of PKC on beta ARs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of the phospholipase C pathway by ATP is mediated exclusively through nucleotide type P2-purinoceptors in C2C12 myotubes.

1. The presence of a nucleotide receptor and a discrete ATP-sensitive receptor on C2C12 myotubes has been shown by electrophysiological experiments. In this study, the ATP-sensitive receptors of C2C12 myotubes were further characterized by measuring the formation of inositol(1,4,5)trisphosphate (Ins(1,4,5)P3) and internal Ca2+. 2. The nucleotides ATP and UTP caused a concentration-dependent increase in Ins(1,4,5)P3 content with comparable time courses (EC50: ATP 33 +/- 2 microM, UTP 80 +/- 4 microM). ADP was less effective in increasing Ins(1,4,5)P3 content of the cells, while selective agonists for P1-, P2X- and P2Y-purinoceptors, adenosine, alpha,beta-methylene ATP and 2-methylthio ATP, appeared to be ineffective. 3. Under Ca(2+)-free conditions, the basal level of Ins(1,4,5)P3 was lower than in the presence of Ca2+, and the ATP- and UTP-induced formation of Ins(1,4,5)P3 was diminished. 4. The Ins(1,4,5)P3 formation induced by optimal ATP and UTP concentrations was not additive. ATP- and UTP-induced Ins(1,4,5)P3 formation showed cross-desensitization, whereas cross-desensitization was absent in responses elicited by one of the nucleotides and bradykinin. 5. The change in Ins(1,4,5)P3 content induced by effective nucleotides was inhibited by suramin. Schild plots for suramin inhibition of Ins(1,4,5)P3 formation in ATP- and UTP-stimulated myotubes showed slopes greater than unity (1.63 +/- 0.09 and 1.37 +/- 0.11, respectively). Apparent pA2 values were 4.50 +/- 0.48 and 4.41 +/- 0.63 for ATP and UTP, respectively. 6. Stimulation of the cells with ATP or UTP induced a rapid increase in intracellular Ca2+, followed by a slow decline to basal levels. Ca2+ responses reached lower maximal values and did not show the slow phase in the absence of extracellular Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two different P2Y receptors linked to steroidogenesis in bovine adrenocortical cells

Both extracellular adenosine 5'-triphosphate (ATP) and uridine 5'-triphosphate (UTP) induced corticoid production (steroidogenesis) concentration-dependently in bovine adrenocortical cells (BA cells). Pertussis toxin (PTX, approx. 2 microg/ml) partially inhibited (approx. 55% inhibition) extracellular ATP (100 microM)-induced steroidogenesis in BA cells. However, PTX did not inhibit extracellular UTP (100 microM)-induced steroidogenesis. Both ATP- and UTP-induced steroidogeneses were significantly inhibited by suramin (50-200 microM). These effects were inhibited significantly by reactive blue-2 (more than 100 microM) and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (more than 100 microM). Both nucleotides (1-100 microM) induced inositol phosphates accumulation and intracellular Ca2+ mobilization, but PTX did not inhibit them. The RT-PCR procedure identified only P2Y2-receptor mRNA in BA cells. These results suggest that extracellular ATP induces steroidogenesis via a unique P2 receptor linked to PTX-sensitive guanine nucleotide-binding protein (G-protein), while extracellular UTP induces steroidogenesis via P2 receptor linked to PTX-insensitive G-protein. Thus, it was concluded that at least two different P2Y-like receptors linking to steroidogenesis exist in BA cells.     

Radioligand binding to muscarinic receptors of bovine aortic endothelial cells

1. Muscarinic receptors on endothelial cells of bovine thoracic aorta were characterized by binding assays in which (-)-[3H]-N-methyl quinuclidinyl benzilate ([3H]-NMeQNB) was used as radioligand. 2. Binding of [3H]-NMeQNB to crude membranes of freshly isolated endothelial cells was atropine-displaceable and of high affinity (KD = 0.48 nM) to a single class of sites (maximum binding capacity: 14 +/- 3 fmol mg-1 protein). Stereospecificity of the binding sites was demonstrated in experiments in which [3H]-NMeQNB binding was inhibited by dexetimide in the nanomolar range (KI = 0.63 nM) and by levetimide, its stereoisomer in the micromolar range (KI = 3.2 microM) (selectivity factor: approximately 5000). 3. Drug competition curves indicated a single class of binding sites for antagonists and the following apparent affinities (KI, nM): methyl atropine: 1.1: 4-diphenylacetoxy N-methyl piperidine methyl bromide (4-DAMP): 3.4; pirenzepine: 16; 11-[2-diethylamino-methyl)-1-piperidinyl- acetyl]-5,11-dihydro-6H-pyrido(2,3-b)1,4-benzodiazepine-6-one (AF-DX 116); 2.500. Competition of acetylcholine with [3H]-NMeQNB was best described by two affinity sites (or states) (KH = 0.82 microM, KL = 1.6 microM). In the presence of guanylimido diphosphate [Gpp(NH)p] (100 microM), acetylcholine affinity (IC50) was slightly, but significantly reduced (factor approximately 4). 4. Binding of [3H]-NMeQNB to freshly harvested intact cells was also atropine-displaceable, stereospecific (selectivity factor: approximately 3500) and of high affinity (KD = 0.35 nM). The maximum binding capacity (9 +/- 2 fmol mg-1 total cell protein) was comparable to that of membranes and corresponded to approximately 900 binding sites per endothelial cell.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein kinase C epsilon-dependent pathway of extracellular signal-regulated protein kinase activation by P2Y1 and P2Y2 purinoceptors that activate cytosolic phospholipase A2 in endothelial cells.

The aim of this study was to investigate the stimulating effects on arachidonic acid release of P2Y1 and P2Y2 receptor-selective agonists, 2-methylthio-ATP (2MeSATP) and UTP, respectively, in bovine pulmonary artery endothelial cells. Exposure of cells to 2MeSATP and UTP led to the release of arachidonic acid, a response which was abolished by the removal of extracellular Ca2+ and methyl arachidonyl fluorophosphonate. Phorbol 12-myristate 13-acetate (PMA) itself not only stimulated arachidonic acid release but also played a permissive role in the response to UTP. However, PMA failed to enhance the arachidonic acid response induced by 2MeSATP, probably due to greater attenuation of the [Ca2+]i increase caused by 2MeSATP than UTP. Inhibition of protein kinase C with Ro 31-8220 (1-[3-(amidinothio) propyl-1H-indoyl-3-yl]-3-(1-methyl-1H-indoyl-3-yl)-maleimide -methane sulphate) and staurosporine, but not with Go 6976 (12-(-2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-indolo(2, 3-a)pyrrolo(3,4-c)carbazole), reduced the arachidonic acid response of 2MeSATP, UTP and PMA. PMA-induced potentiation of the UTP response reached a maximum after a 1-h preincubation, then declined and eventually lost its effect when the preincubation lasted up to 8 h. Among the protein kinase C isoforms present in endothelial cells, betaI and epsilon could be down-regulated by treatment with PMA for 4-24 h. PD 098059 (2-(2-Amino-3-methoxyphenyl)-4H-1-benzopyran-4-one) inhibited extracellular signal-regulated protein kinase activation, cytosolic phospholipase A2 phosphorylation and arachidonic acid release caused by 2MeSATP, UTP and PMA. Taken together, our results demonstrate that P2Y1 and P2Y2 purinoceptors mediate arachidonic acid release by activating cytosolic phospholipase A2 through an elevation of [Ca2+]i and protein kinase C epsilon-, extracellular signal-regulated protein kinase-dependent phosphorylation.     

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

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

Competitive regulation of phospholipase C responses by cAMP and calcium.

DDT1-MF2 smooth muscle cells demonstrated a robust phospholipase C response to norepinephrine, as detected by inositol phosphate accumulation. A selective A1-adenosine receptor agonist, cyclopentyladenosine, caused only a minor stimulation of phospholipase C, which was eliminated in the absence of added extracellular calcium. The simultaneous addition of norepinephrine and cyclopentyladenosine resulted in a synergistic increase in phosphoinositide hydrolysis either in the absence or in the presence of external calcium. In the presence of external calcium and a calcium ionophore, and adenosine agonist caused a significant stimulation of phosphoinositide hydrolysis without the addition of norepinephrine. Influx of extracellular calcium through voltage-sensitive calcium channels did not appear to be required to observe an effect of cyclopentyladenosine, because neither calcium channel antagonists (nifedipine, verapamil, and LaCl3) nor a chelator of extracellular calcium (ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid) were able to alter the degree of potentiation of norepinephrine-stimulated phosphoinositide hydrolysis due to the adenosine agonist. On the other hand, buffering of intracellular calcium concentration with the membrane-permeant calcium chelator quin2 blocked the potentiation. This blockade of potentiation by quin2 was reversed by the addition of extracellular calcium. Agents that stimulated cAMP production or membrane-permeable analogues of cAMP also blocked the action of the adenosine agonist to potentiate norepinephrine-stimulated phosphoinositide hydrolysis. This effect of cAMP was less pronounced in the presence of elevated extracellular calcium and was abolished in the presence of a calcium ionophore. When norepinephrine-stimulated calcium transients were quantitated using fura-2 fluorescence, a reduction in the amplitude of the calcium response was observed in the presence of forskolin. Conversely, both the amplitude and the duration of the calcium response were enhanced by the addition of the adenosine agonist. The results of these studies suggest that the mechanism by which adenosine receptors enhance the stimulation of phosphoinositide hydrolysis is dependent upon a rise in intracellular Ca2+ concentration resulting from the simultaneous activation of alpha 1-adrenergic receptors. The results further suggest that cAMP inhibits this mechanism by decreasing the norepinephrine-stimulated rise in intracellular Ca2+ concentration.     

P2Y receptors of MDCK cells: epithelial cell regulation by extracellular nucleotides

1. Madin-Darby canine kidney (MDCK) cells, a well- differentiated renal epithelial cell line derived from distal tubule/collecting duct, respond to extracellular nucleotides by altering ion flux and the production of arachidonic acid-derived products, in particular prostaglandin E2 (PGE2). Our work has defined the receptors and signalling events involved in such responses. 2. We have found evidence for expression of at least three P2Y receptor subtypes (P2Y1, P2Y2 and P2Y11) in MDCK-D1 cells, a subclone from parental MDCK. 3. These receptors appear to couple to increases in calcium and protein kinase C activity, probably via a Gq/G11-mediated activation of phospholipase C. 4. In addition, P2Y receptor activation can promote a prominent increase in cAMP. This includes both a P2Y2 receptor-mediated cyclo-oxygenase (COX)-dependent component and another COX-independent component mediated by other P2Y receptors. 5. We have documented that changing media in which cells are grown releases ATP and, in turn, activates P2Y receptors. Such release of ATP contributes in a major way to basal cAMP levels in these cells. 6. The data indicate that MDCK cells are a useful model to define the regulation of epithelial cells by extracellular nucleotides. Of particular note, spontaneous or stretch-induced release of ATP and subsequent activation of one or more P2Y receptors contributes to establishing the basal activity of signalling pathways.