Atherosclerosis-related remodeling of aortic relaxation to purines in the Watanabe heritable hyperlipidemic rabbit

The mechanism of the unimpaired relaxant effect of ATP in the Watanabe heritable hyperlipidemic rabbit aorta was investigated to elucidate the involvement of P2y purinoceptor at the endothelial level during atherosclerosis. Experiments were carried out on isolated thoracic aorta from such rabbits that were 12 months of age. The potent P2y purinoceptor agonist, 2-methylthio-ATP, did not induce any endothelium- or smooth muscle-dependent relaxation, thus excluding any involvement by the P2y purinoceptor. ADP, but not AMP, produced relaxation of the aorta by acting at both endothelial and smooth muscle levels. Adenosine relaxed the vessel by acting only in smooth muscle. The maintained endothelial relaxant effect of ATP and ADP is therefore not due to activation of P1 or P2y purinoceptors but may involve activation of a remodeled purinergic receptor site that emerges with the progression of atherosclerosis. This site is antagonized by methylene blue. The disorganization of the endothelial monolayer observed in the morphological analysis may be related to functional remodeling of the endothelial purinergic activity in atherosclerosis.     

Types of purinoceptors and phospholipase A2 involved in the activation of the platelet-activating factor-dependent transacetylase activity and arachidonate release by ATP in endothelial cells

Acyl analogs of PAF are the major products synthesized during agonist stimulation of endothelial cells. We have previously shown that PAF: 1-acyl-2-lyso-sn-glycero-3-phosphocholine transacetylase in calf pulmonary artery endothelial cells is activated by ATP through protein phosphorylation, and the increase in transacetylase activity by ATP contributes to the biosynthesis of acyl analogs of PAF (J. Biol. Chem. 272, 17431-17437, 1997). To understand the mechanisms(s) by which ATP stimulates acyl analogs of PAF production, we have identified the subtypes of the purinergic receptor that are linked to the activation of two enzymes involved in the generation of acyl analogs of PAF, namely, transacetylase and phospholipase A2. Experiments with transient transfection of the cells with antisense and sense thio-oligonucleotide to cytosolic phospholipase A2 (cPLA2) were also performed to evaluate whether downstream activation of cPLA2 is involved in ATP-receptor mediated induction of arachidonate release and synthesis of radylacetyl-GPC. We found that the P2u/P2Y2 receptor, which recognizes a pyrimidine nucleotide, UTP, as well as purine nucleotides, shows a potency profile of UTP > ATP = ATP gamma S > 2-methylthio-ATP in mediating the activation of PAF: lysophospholipid transacetylase. On the other hand, ADP beta S and 2-methylthio-ATP have similar potencies as ATP but have lower potencies than UTP and ATP gamma S in stimulating the release of arachidonate. These results suggest that both P2u/P2Y2 and P2y/P2Y1 receptor subtypes promote arachidonate release. In addition, transient transfection of endothelial cells with cPLA2 antisense but not the sense thio-oligonucleotide inhibited the stimulation of arachidonate release and [3H]acetate incorporation into radyl[3H]acetyl-GPC. Thus, our data suggest that a receptor-mediated process is involved in the activation of transacetylase for the induced synthesis of acyl analogs of PAF in endothelial cells. Furthermore, it is likely that cPLA2 supplies the lysophospholipids as substrates for the transacetylation reaction.     

P2 receptor modulation of voltage-gated potassium currents in Brown adipocytes

Using patch voltage-clamp techniques, we find there are two components to the voltage-gated potassium current (IKv) in rat brown adipocytes. The components differ in their gating and responses to purinergic stimulation, but not their pharmacology. IKv-A recovers from inactivation at physiological membrane potentials, while IKv-B inactivation recovers at more negative potentials. Both currents are >90% blocked by similar concentrations of quinine and tetraethylammonium, but not by beta-dendrotoxin, charybdotoxin, or apamin. The two current components are differentially modulated by extracellular ATP. ATP shifts the voltage dependence of IKv-A inactivation negative by 38 +/- 5 mV (n = 35, +/-SEM) and shifts activation by -14 +/- 2 mV in whole-cell experiments. ATP did not affect the steady state inactivation voltage dependence of IKv-B, but did apparently convert IKv-A into IKv-B. The pharmacology of the inactivation shift is consistent with mediation by a P2 purinergic receptor. Purinergic stimulation of perforated-patch clamped cells causes hyperpolarizing shifts in the window current of IKv-A by shifting inactivation -18 +/- 4 mV and activation -7 +/- 2 mV (n = 16). Since perforated-patch recordings will most closely resemble in vivo cell responses, this ATP-induced shift in the window current may facilitate IKv activation when the cell depolarizes. IKv activity is necessary for the proliferation and differentiation of brown adipocytes in culture (Pappone, P.A., and S.I. Ortiz-Miranda. 1993. Am. J. Physiol. 264:C1014-C1019) so purinergic modulation of IKv may be important in altering adipocyte growth and development.     

Functional characterisation of P2 purinoceptors in PC12 cells by measurement of radiolabelled calcium influx

The aim of this study was to determine whether 45Ca2+ influx could be used as a quantitative measure of channel activation for functional characterisation of P2X purinoceptors in cell lines. In undifferentiated PC12 cells, grown in suspension, ATP (EC50 = 45 microM), ATP gamma S (EC50 = 50 microM) and 2-meSATP (EC50 = 81 microM) but not alpha beta meATP (1 mM) stimulated 45Ca2+ influx 2-5 fold. This effect did not appear to be due to activation of P2U or P2Y purinoceptors since 1 mM UTP, ADP or ADP beta S did not produce any significant effect. Similarly, the effects of ATP were not apparently mediated through activation of P2Z purinoceptors since dibenzylATP behaved as a weak (EC50 = 191 microM) partial agonist (Maximal effect 29.5% of ATP maximum) and there was no detectable ATP-stimulated ethidium bromide uptake in the PC12 cells. ATP-stimulated 45Ca2+ influx was not affected by nifedipine suggesting that it was not secondary to activation of L-type calcium channels and rather reflected influx through a P2X purinoceptor present in these cells. The ATP-stimulated 45Ca2+ influx could be reduced by monovalent cations, presumably as a result of direct competition for influx through the cation channel, with the following rank order of potency:- guanidinium (EC50 = 16 mM) > sodium > Tris > choline > N-methyl-D-glucamine = sucrose). A number of P2 purinoceptor antagonists inhibited ATP-stimulated 45Ca2+ influx. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (3-300 microM), pyridoxal 5-phosphate (3-300 microM) and d-tubocurarine (30-300 microM) produced an insurmountable antagonism of responses to ATP, with no marked change in agonist EC50. Suramin (100-300 microM) and cibacron blue (30-300 microM) produced a surmountable antagonism while DIDS (4,4'-diisothiocyanatostilbene-2,2'disulfonic acid) only antagonised responses to ATP at concentrations in excess of 300 microM. The general properties of the P2X purinoceptor population identified in these cells were consistent with them being P2X2 purinoceptors. These findings suggest that ATP-stimulated 45Ca2+ influx may be used as a reliable and quantitative functional assay for characterisation of P2X purinoceptor subtypes in cell lines.     

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.     

Value of 3D CT in diagnosis and treatment of fractures of the tibial plateau]

Adenosine triphosphate (ATP) is a signaling molecule for brain cells including astrocytes. In these cells, it has been shown that ATP stimulates myelin basic protein (MBP) kinase activity which is believed to represent the Erk family of MAP kinases. Indeed, we show that ATP activates simultaneously MBP kinase activity and phosphotyrosine incorporation in p42 Erk2 and p44 Erk1. Maximal effect of ATP is obtained at 50 microM after 5 min and disappears after 60 min. Effect of ATP is mimicked by 2-methylthio-ATP whereas alpha beta-methyleneadenosine 5' triphosphate (AMP-CPP) and adenosine do not promote any effect. Uridine triphosphate (UTP) activates also p42 and p44 MAP kinases. These observations indicate that p42-p44 MAP kinases activation can be obtained through P2v and P2u receptors. Purinergic stimulation of Erk is insensitive to pertussis toxin which inactivates heterotrimeric Gi protein. It is not inhibited by a PLA2 inhibitor (4 bromophenacyl bromide [B phi B]) and the PI3 kinase inhibitor, wortmannin. In contrast, purinergic stimulation of Erk is partially inhibited by the PKC inhibitor. GF109203X, at 5 microM and suppressed when extracellular calcium is complexed by ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA).     

Apyrase administration prolongs discordant xenograft survival

Platelet thrombi and vascular inflammation are prominent features of discordant xenograft rejection. The purinergic nucleotides ATP and ADP, which are secreted from platelets and released by injured endothelial cells (EC), are important mediators of these reactions. Quiescent EC express the ectoenzyme ATP-diphosphohydrolase (ATPDase; an apyrase), which exerts an important thromboregulatory function by hydrolyzing both ATP and ADP. We have shown that ATPDase activity is rapidly lost from the surface of the EC following ischemia-reperfusion injury and during xenograft rejection. The aim of this study was to supplement ATPDase activity within xenografts by infusion of soluble apyrases, and thereby validate the importance of local ATPDase activity in the modulation of xenograft rejection. Lewis rats underwent heterotopic cardiac xenografting from guinea pigs and apyrase was administered intravenously (200 U/kg) as a single dose to evaluate effects on hyperacute rejection (HAR). This initial dose was followed by a continuous apyrase infusion (8.0 U/kg/hr) directly into the graft aorta in combination with systemic cobra venom factor (CVF) administration to deplete complement when delayed xenograft rejection (DXR) was studied. Functional apyrase levels in vivo were assessed by the capacity of blood samples taken at the time of surgery and rejection to inhibit platelet aggregation in vitro. Apyrase administration significantly prolonged graft survival in HAR and DXR. Functional assays showed inhibition of platelet aggregation suggesting effective systemic antiaggregatory effects of the administered apyrases. Histologic studies showed that apyrase administration abrogated local platelet aggregation and activation in HAR and DXR. Our data demonstrate that local administration of apyrase prolonged discordant xenograft survival. These observations emphasize the potential importance of purinergic mediators in platelet activation during xenograft rejection.     

ATP-stimulated activation of the mitogen-activated protein kinases through ionotrophic P2X2 purinoreceptors in PC12 cells. Difference in purinoreceptor sensitivity in two PC12 cell lines

Extracellular purine nucleotides elicit a diverse range of biological responses through binding to specific cell surface receptors. The ionotrophic P2X subclass of purinoreceptors respond to ATP by stimulation of calcium ion permeability; however, it is unknown how P2X purinoreceptor activation is linked to intracellular signaling pathways. We report that stimulation of PC12 cells with ATP results in the activation of the mitogen-activated protein (MAP) kinases ERK1 and ERK2 and was wholly dependent upon extracellular calcium ions. Treatment of the cells with adenosine, AMP, ADP, UTP, or alpha,beta-methylene ATP was without effect; however, MAP kinase activation was abolished by pretreatment with suramin and reactive blue 2. The calcium-activated tyrosine kinase, Pyk2, acts as an upstream regulator of the MAP kinases and became tyrosine phosphorylated following treatment of the cells with ATP. We have ruled out the involvement of depolarization-mediated calcium influx because specific blockers of voltage-gated calcium channels did not affect MAP kinase activation. These data provide direct evidence that calcium influx through P2X2 receptors results in the activation of the MAP kinase cascade. Finally, we demonstrate that a different line of PC12 cells respond to ATP through P2Y2 purinoreceptors, providing an explanation for the conflicting findings of purine nucleotide responsiveness in PC12 cells.     

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.     

Pulsatile stretch stimulates superoxide production and activates nuclear factor-kappa B in human coronary smooth muscle

There is increasing evidence that oxidative stress is of pathophysiological importance in cardiovascular disease. Mechanical forces such as pulsatility may also contribute. Using human coronary artery smooth muscle cells (HCAS), we tested the hypothesis that stretch-induced cell proliferation is associated with oxidative stress. Stretch induced DNA synthesis in HCAS, and this was prevented by the antioxidants N-acetylcysteine and pyrrolidinedithiocarbamate (PDTC). Pulsatile stretch also increased superoxide production from HCAS in a time- and stretch dependent manner. Stretch-induced superoxide production was inhibited by diphenyleneiodoniumchloride, an NADPH oxidase inhibitor, and p-chloromercuriphenylsulfonic acid, an NADH oxidase inhibitor, but not by the xanthine oxidase inhibitor oxypurinol or the cyclooxygenase inhibitor indomethacin. In electrophoretic mobility shift assays, tumor necrosis factor-alpha activated nuclear factor-kappa B (NF-kappa B) with a peak at approximately 3 hours, whereas pulsatile stretch showed sustained activation during stimulation for up to 24 hours. The sustained activation of NF-kappa B was abolished by cotreatment with N-acetylcysteine or PDTC. Furthermore, treatment of HCAS with antisense p65 and p50 oligodeoxynucleotides of NF-kappa B inhibited stretch-induced DNA synthesis. We propose that pulsatile stretch increases oxidative stress and, in turn, promotes DNA synthesis via NF-kappa B in cultured human coronary artery smooth muscle cells.