Regulation of transcervical permeability by two distinct P2 purinergic receptor mechanisms

Micromolar concentrations ofATP stimulate biphasic change in transepithelial conductance acrossCaSki cultures, an acute increase (phase I response) followed by aslower decrease (phase II response). Phase I andphase II responses involve two distinct calcium-dependentpathways, calcium mobilization and calcium influx. To test thehypothesis that phase I and phase II responsesare mediated by distinct P2 purinergic receptors, changes inpermeability were uncoupled by blocking calcium mobilization with1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid(BAPTA) or by lowering extracellular calcium, respectively. Under theseconditions ATP EC₅₀ was 25 µM for phase Iresponse and 2 µM for phase II response. The respectiveagonist profiles were ATP > UTP > adenosine5'-O-(3-thiotriphosphate) (ATP-S) = N⁶-([6-aminohexyl]carbamoylmethyl)adenosine5'-triphosphate (A8889) > GTP and UTP > ATP > GTP = A8889 > ATP-S. Suramin blocked phase Iresponse and ATP-induced calcium mobilization, whereas pyridoxal phosphate-6-azophenyl-2',4-disulfonic acid (PPADS) blocked phase II response and ATP-augmented calcium influx. ATP time course andpharmacological profiles for phase II response and augmented calcium influx were similar, with a time constant of 2 min and asaturable concentration-dependent effect (EC₅₀ of 2-3µM). RT-PCR experiments revealed expression of mRNA for both theP2Y₂ and P2X₄ receptors. These results suggestthat the ATP-induced phase I and phase IIresponses are mediated by distinct P2 purinergic receptor mechanisms.

     

Regulation of P2X7-induced pore formation and cell death in pericyte-containing retinal microvessels

The purpose if this study was to elucidate how extracellular ATP causes cell death in the retinal microvasculature. Although ATP appears to serve as a vasoactive signal acting via P2X₇ and P2Y₄ purinoceptors, this nucleotide can kill microvascular cells of the retina. Because P2X₇ receptor activation causes transmembrane pores to form and microvascular cells to die, we initially surmised that pore formation accounted for ATP's lethality. To test this hypothesis, we isolated pericyte-containing microvessels from rat retinas, assessed cell viability using Trypan blue dye exclusion, detected pores by determining the uptake of the fluorescent dye YO-PRO-1, measured intracellular Ca²⁺ with the use of fura-2, and monitored ionic currents via perforated patch pipettes. As predicted, ATP-induced cell death required P2X₇ receptor activation. However, we found that pore formation was minimal because ATP's activation of P2Y₄ receptors prevented P2X₇ pores from forming. Rather than opening lethal pores, ATP kills via a mechanism involving voltage-dependent Ca²⁺ channels (VDCC). Our experiments suggest that when high concentrations of ATP caused nearly all microvascular P2X₇ receptor channels to open, the resulting profound depolarization opened VDCC. Consistent with lethal Ca²⁺ influx via VDCC, ATP-induced cell death was markedly diminished by the VDCC blocker nifedipine or a nitric oxide (NO) donor that inhibited microvascular VDCC. We propose that purinergic vasotoxicity is normally prevented in the retina by NO-mediated inhibition of VDCC and P2Y₄-mediated inhibition of P2X₇ pore formation. Conversely, dysfunction of these protective mechanisms may be a previously unrecognized cause of cell death within the retinal microvasculature. calcium channels; capillaries; purinoceptors; vasotoxicity     

ATP activates DNA synthesis by acting on P2X receptors in human osteoblast-like MG-63 cells

In human osteoblast-like MG-63cells, extracellular ATP increased [³H]thymidineincorporation and cell proliferation and synergistically enhancedplatelet-derived growth factor- or insulin-like growth factor I-induced[³H]thymidine incorporation. ATP-induced[³H]thymidine incorporation was mimicked by thenonhydrolyzable ATP analogs adenosine5'-O-(3-thiotriphosphate) and adenosine 5'-adenylylimidodiphosphate and was inhibited by the P2purinoceptor antagonist suramin, suggesting involvement of P2purinoceptors. The P2Y receptor agonist UTP and UDP and a P2Y receptorantagonist reactive blue 2 did not affect [³H]thymidineincorporation, whereas the P2X receptor antagonist pyridoxalphosphate-6-azophenyl-2',4-disulfonic acid inhibited ATP-induced[³H]thymidine incorporation, suggesting that ATP-inducedDNA synthesis was mediated by P2X receptors. RT-PCR analysis revealedthat MG-63 cells expressed P2X₄, P2X₅,P2X₆, and P2X₇, but not P2X₁,P2X₂, and P2X₃, receptors. In fura 2-loadedcells, not only ATP, but also UTP, increased intracellularCa²⁺ concentration, and inhibitors for severalCa²⁺-activated protein kinases had no effect on ATP-inducedDNA synthesis, suggesting that an increase in intracellularCa²⁺ concentration is not indispensable for ATP-induced DNAsynthesis. ATP increased mitogen-activated protein kinase activity in aCa²⁺-independent manner and synergistically enhancedplatelet-derived growth factor- or insulin-like growth factor I-inducedkinase activity. Furthermore, the mitogen-activated protein kinasekinase inhibitor PD-98059 totally abolished ATP-induced DNA synthesis. We conclude that ATP increases DNA synthesis and enhances the proliferative effects of growth factors through P2X receptors byactivating a mitogen-activated protein kinase pathway.

     

Expression of P2X(7) purinoceptors on human lymphocytes and monocytes: evidence for nonfunctional P2X(7) receptors

Lymphocytes from normal subjects and patients with B-chroniclymphocytic leukemia (B-CLL) show functional responses to extracellular ATP characteristic of the P2X₇ receptor (previously termedP2Z). These responses include opening of a cation-selectivechannel/pore that allows entry of the fluorescent dye ethidium andactivation of a membrane metalloprotease that sheds the adhesionmolecule L-selectin. The surface expression of P2X₇receptors was measured in normal leucocytes, platelets, and B-CLLlymphocytes and correlated with their functional responses. Monocytesshowed four- to fivefold greater expression of P2X₇ than B,T, and NK lymphocytes, whereas P2X₇ expression onneutrophils and platelets was weak. All cell types demonstratedabundant intracellular expression of this receptor. All 12 subjectswith B-CLL expressed lymphocyte P2X₇ at about the samelevel as B lymphocytes from normal subjects. P2X₇ function, measured by ATP-induced uptake of ethidium, correlated closely withsurface expression of this receptor in normal and B-CLL lymphocytes andmonocytes (n = 47, r = 0.70; P< 0.0001). However, in three patients the ATP-induced uptake ofethidium into the malignant B lymphocytes was low or absent. The lackof P2X₇ function in these B lymphocytes was confirmed bythe failure of ATP to induce Ba²⁺ uptake into theirlymphocytes. This lack of function of the P2X₇ receptorresulted in a failure of ATP-induced shedding of L-selectin, anadhesion molecule that directs the recirculation of lymphocytes fromblood into the lymph node.

     

Glu496Ala polymorphism of human P2X7 receptor does not affect its electrophysiological phenotype

A glutamateto alanine exchange at amino acid position 496 of the humanP2X₇ receptor was recently shown to be associated with aloss of function in human B lymphocytes in terms of ATP-induced ethidium⁺ uptake, Ba²⁺ influx, and induction ofapoptosis (Gu BJ, Zhang WY, Worthington RA, Sluyter R, Dao-UngP, Petrou S, Barden JA, and Wiley JS. J Biol Chem 276:11135-11142, 2001). Here we analyzed the effect of theGlu⁴⁹⁶ to Ala exchange on the channel properties of thehuman P2X₇ receptor expressed in Xenopus oocyteswith the two-microelectrode voltage-clamp technique. The amplitudes ofATP-induced whole cell currents characteristic of functionalexpression, kinetic properties including ATP concentration dependence,and permeation behavior were not altered by this amino acid exchange.Also in HEK293 cells, the Ala⁴⁹⁶ mutant mediated typicalP2X₇ receptor-dependent currents like the parentGlu⁴⁹⁶ hP2X₇ receptor. Because the function ofthe P2X₇ receptor as an ATP-gated channel for small cationsincluding Ba²⁺ remained unaffected by this mutation, weconclude that Glu⁴⁹⁶ plays a critical role in poreformation but does not determine the ion channel properties of thehuman P2X₇ receptor.

     

Polarized expression of human P2Y receptors in epithelial cells from kidney, lung, and colon

Eight human G protein-coupled P2Y receptors (P2Y₁, P2Y₂, P2Y₄, P2Y₆, P2Y₁₁, P2Y₁₂, P2Y₁₃, and P2Y₁₄) that respond to extracellular nucleotides have been molecularly identified and characterized. P2Y receptors are widely expressed in epithelial cells and play an important role in regulating epithelial cell function. Functional studies assessing the capacity of various nucleotides to promote increases in short-circuit current (I_sc) or Ca²⁺ mobilization have suggested that some subtypes of P2Y receptors are polarized with respect to their functional activity, although these results often have been contradictory. To investigate the polarized expression of the family of P2Y receptors, we determined the localization of the entire P2Y family after expression in Madin-Darby canine kidney (MDCK) type II cells. Confocal microscopy of polarized monolayers revealed that P2Y₁, P2Y₁₁, P2Y₁₂, and P2Y₁₄ receptors reside at the basolateral membrane, P2Y₂, P2Y₄, and P2Y₆ receptors are expressed at the apical membrane, and the P2Y₁₃ receptor is unsorted. Biotinylation studies and I_sc measurements in response to the appropriate agonists were consistent with the polarized expression observed in confocal microscopy. Expression of the G_q-coupled P2Y receptors (P2Y₁, P2Y₂, P2Y₄, P2Y₆, and P2Y₁₁) in lung and colonic epithelial cells (16HBE14o– and Caco-2 cells, respectively) revealed a targeting profile nearly identical to that observed in MDCK cells, suggesting that polarized targeting of these P2Y receptor subtypes is not a function of the type of epithelial cell in which they are expressed. These experiments highlight the highly polarized expression of P2Y receptors in epithelial cells. Madin-Darby canine kidney; 16HBE14o–; Caco-2; confocal microscopy; polarized targeting     

Retinoids modulate P2U purinergic receptor-mediated changes in transcervical paracellular permeability

In human cervical cells, extracellular ATP induces an acutedecrease in the resistance of the lateral intercellular space, thephase I response, followed by adelayed increase in tight junctional resistance, thephase II response. These responses depend on vitamin A because incubation of cells in retinoid-free medium(RFM) abolished both responses. Treatment with retinoic acid restoredthe phase I response in full, but theamplitude of the phase II response wasrestored only partly. Shorter incubations and lower concentrations ofretinoic acid [half-maximal effective concentration(K_1/2) = 0.1 µM] were required for restoring the phaseI response than were required for reversing thephase II response(K_1/2 = 1 µM).The phase I response could be restoredby ligands that bind to either retinoic acid receptors (RARs) orretinoid X receptors, but only RAR agonists had an effect onphase II response. RFM had no effecton decreases in resistance induced by ionomycin, but it attenuatedphase II-like increases in resistanceinduced by KCl or by1,2-dioctanoyl-sn-diglycerol (diC8).Actinomycin D blocked phase IIresponse but not phase I response orthe responses to ionomycin, KCl, or diC8. These results suggest thatretinoids act on cervical cells via distinct retinoid receptormechanisms and modulate phase I andphase II changes in resistance byregulating distinct signal mechanisms.

     

Maitotoxin and P2Z/P2X7 purinergic receptor stimulation activate a common cytolytic pore

The effects ofmaitotoxin (MTX) on plasmalemma permeability are similar to thosecaused by stimulation of P2Z/P2X₇ionotropic receptors, suggesting that1) MTX directly activatesP2Z/P2X₇ receptors or2) MTX andP2Z/P2X₇ receptor stimulationactivate a common cytolytic pore. To distinguish between these twopossibilities, the effect of MTX was examined in1) THP-1 monocytic cells before andafter treatment with lipopolysaccharide and interferon-, a maneuverknown to upregulate P2Z/P2X₇receptor, 2) wild-type HEK cells andHEK cells stably expressing theP2Z/P2X₇ receptor, and3) BW5147.3 lymphoma cells, a cellline that expresses functional P2Z/P2X₇ channels that are poorlylinked to pore formation. In control THP-1 monocytes, addition of MTXproduced a biphasic increase in the cytosolic freeCa²⁺ concentration([Ca²⁺]_i);the initial increase reflects MTX-inducedCa²⁺ influx, whereas the secondphase correlates in time with the appearance of large pores and theuptake of ethidium. MTX produced comparable increases in[Ca²⁺]_iand ethidium uptake in THP-1 monocytes overexpressing theP2Z/P2X₇ receptor. In bothwild-type HEK and HEK cells stably expressing theP2Z/P2X₇ receptor, MTX-inducedincreases in[Ca²⁺]_iand ethidium uptake were virtually identical. The response of BW5147.3cells to concentrations of MTX that produced large increases in[Ca²⁺]_ihad no effect on ethidium uptake. In both THP-1 and HEK cells, MTX- andBz-ATP-induced pores activate with similar kinetics and exhibit similarsize exclusion. Last, MTX-induced pore formation, but not channelactivation, is greatly attenuated by reducing the temperature to22°C, a characteristic shared by theP2Z/P2X₇-induced pore. Together,the results demonstrate that, although MTX activates channels that aredistinct from those activated byP2Z/P2X₇ receptor stimulation, thecytolytic/oncotic pores activated by MTX- and Bz-ATP are indistinguishable.

     

RNA interference targeted to multiple P2X receptor subtypes attenuates zinc-induced calcium entry

A postulated therapeutic avenue in cystic fibrosis (CF) is activation of Ca²⁺-dependent Cl^– channels via stimulation of Ca²⁺ entry from extracellular solutions independent of CFTR functional status. We have shown that extracellular zinc and ATP induce a sustained increase in cytosolic Ca²⁺ in human airway epithelial cells that translates into stimulation of sustained secretory Cl^– transport in non-CF and CF human and mouse airway epithelial cells, cell monolayers, and nasal mucosa. On the basis of these studies, the Ca²⁺ entry channels most likely involved were P2X purinergic receptor channels. In the present study, molecular and biochemical data show coexpression of P2X₄, P2X₅, and P2X₆ subtypes in non-CF (16HBE14o^–) and CF (IB3-1) human bronchial epithelial cells. Other P2X receptor Ca²⁺ entry channel subtypes are expressed rarely or not at all in airway epithelia, epithelial cell models from other CF-relevant tissues, or vascular endothelia. Novel transient lipid transfection-mediated delivery of small interference RNA fragments specific to P2X₄ and P2X₆ (but not P2X₅) into IB3-1 CF human airway epithelial cells inhibited extracellular zinc- and ATP-induced Ca²⁺ entry markedly in fura-2 Ca²⁺ measurements and "knocked down" protein by >65%. These data suggest that multiple P2X receptor Ca²⁺ entry channel subtypes are expressed in airway epithelia. P2X₄ and P2X₆ may coassemble on the airway surface as targets for possible therapeutics for CF independent of CFTR genotype. purinergic receptors; zinc receptors; airway epithelia; cystic fibrosis; therapy     

P2Y purinoceptors induce changes in intracellular calcium in acinar cells of rat lacrimal glands

Adenosine 5′-triphosphate (ATP) is an extracellular signal that regulates various cellular functions. Cellular secretory activities are enhanced by ATP as well as by cholinergic and adrenergic stimuli. The present study aimed to determine which purinoceptors play a role in ATP-induced changes in the intracellular concentration of calcium ions ([Ca²⁺]_i) and in the fine structure of acinar cells of rat lacrimal glands. ATP induced exocytotic structures, vacuolation and an increase in [Ca²⁺]_i in acinar cells. The removal of extracellular Ca²⁺ or the use of Ca²⁺ channel blockers partially inhibited the ATP-induced [Ca²⁺]_i increase. U73122 (an antagonist of PLC) and heparin (an antagonist of IP₃ receptors) did not completely inhibit the ATP-induced [Ca²⁺]_i increase. P1 purinoceptor agonists did not induce any changes in [Ca²⁺]_i, whereas suramin (an antagonist of P2 receptors) completely inhibited ATP-induced changes in [Ca²⁺]_i. A P2Y receptor agonist, 2-MeSATP, induced a strong increase in [Ca²⁺]_i, although UTP (a P2Y_2,4,6 receptor agonist) had no effect, and reactive blue 2 (a P2Y receptor antagonist) resulted in partial inhibition. The potency order of ATP analogs (2-MeSATP > ATP ⋙ UTP) suggested that P2Y₁ played a significant role in the cellular response to ATP. BzATP (a P2X₇ receptor agonist) induced a small increase in [Ca²⁺]_i, but α,β-meATP (a P2X_1,3 receptor agonist) had no effect. RT-PCR indicated that P2X_2,3,4,5,6,7 and P2Y_1,2,4,12,14 are expressed in acinar cells. In conclusion, the response of acinar cells to ATP is mediated by P2Y (especially P2Y₁) as well as by P2X purinoceptors.     

环境因子对角倍蚜秋迁蚜生殖和雌性蚜发育的影响

研究了环境因子对角倍蚜Schlechtendalia chinensis (Bell) 秋迁蚜生殖和雌性蚜发育的影响。温、湿度单因子试验表明,秋迁蚜在26℃和80%RH条件下有最大生殖量;温、湿度对秋迁蚜生殖量的影响均符合开口向下的二次抛物线变化趋势,极端温、湿度会导致生殖量的下降。采用三元一次正交组合设计,研究了环境温度（X₁）、湿度（X₂）和光照强度（X₃）三因子不同水平组合对雌性蚜发育的影响,表明温度是影响发育历期的主要因子,其次是光照强度,最后是湿度。因此,适当高温、强光照条件可以加快雌性蚜发育;而适当高湿条件可以降低雌性蚜的发育速率而延长其发育历期。在人工培育角倍蚜生产中,创造有利于秋迁蚜生殖的温、湿度条件可以使秋迁蚜产下较多的越冬侨蚜;在适当降低温度、增加湿度的阴暗条件下贮留雌性蚜可以适当延长其发育,以使角倍蚜与盐肤木在物候上达到最佳吻合。     

Estimating the capacity for improvement in risk prediction with a marker

Consider a set of baseline predictors X to predict a binaryoutcome D and let Y be a novel marker or predictor. This paperis concerned with evaluating the performance of the augmentedrisk model P(D = 1|Y,X) compared with the baseline model P(D= 1|X). The diagnostic likelihood ratio, DLR_X(y), quantifiesthe change in risk obtained with knowledge of Y = y for a subjectwith baseline risk factors X. The notion is commonly used inclinical medicine to quantify the increment in risk predictiondue to Y. It is contrasted here with the notion of covariate-adjustedeffect of Y in the augmented risk model. We also propose methodsfor making inference about DLR_X(y). Case–control studydesigns are accommodated. The methods provide a mechanism toinvestigate if the predictive information in Y varies with baselinecovariates. In addition, we show that when combined with a baselinerisk model and information about the population distributionof Y given X, covariate-specific predictiveness curves can beestimated. These curves are useful to an individual in decidingif ascertainment of Y is likely to be informative or not forhim. We illustrate with data from 2 studies: one is a studyof the performance of hearing screening tests for infants, andthe other concerns the value of serum creatinine in diagnosingrenal artery stenosis.     

影响叶螨磷酸酯酶活性的四因子数学模型

应用二次回归通用旋转组合设计,组建了影响叶螨磷酸酯酶（酸性和碱性）活性的四因子（缓冲液Ph值X₁、温浴时间X₂、反应温度X₃、底物浓度X₄）数学模型: Y_酸性=0.456380+0.107889X₂+0.069027X₃-0.026836X₁²-0.030794X₃², F=24.98,P<0.01;Y_碱性=0.267286-0.200736X₁+0.049541X₂+0.030930X₃-.049063X₁X₂+0.053585X₁²-0.049665X₂^2, F=57.68,P<0.01。结果表明,温浴时间是影响叶螨酸性磷酸酯酶活性的关键因子,在缓冲液pH 4.4、底物浓度8.5×10^-3 mol/L、42℃温浴40 min测得该酶活性最强。影响碱性磷酸酯酶活性的关键因子则是缓冲液pH值,pH 9.0、37℃恒温30 min、底物7.5×10^-3 mol/L的条件下,光密度值最大。两种酶的最大吸收峰波长为405 nm。     

Mechanisms of P2X7 receptor-mediated ERK1/2 phosphorylation in human astrocytoma cells

Astrocytes are involved in normal andpathological brain functions, where they become activated and undergoreactive gliosis. Astrocytes have been shown to respond toextracellular nucleotides via the activation of P2 receptors, either Gprotein-coupled P2Y receptors or P2X receptors that are ligand-gatedion channels. In this study, we have examined the manner in whichactivation of the P2X₇ nucleotide receptor, anextracellular ATP-gated ion channel expressed in astrocytes, can leadto the phosphorylation of ERK1/2. Results showed that theP2X₇ receptor agonist2',3'-O-(4-benzoyl)benzoyl-ATP induced ERK1/2phosphorylation in human astrocytoma cells overexpressing therecombinant rat P2X₇ receptor (rP2X₇-R), aresponse that was inhibited by the P2X₇ receptorantagonist, oxidized ATP. Other results suggest thatrP2X₇-R-mediated ERK1/2 phosphorylation was linked to thephosphorylation of the proline-rich/Ca²⁺-activated tyrosinekinase Pyk2, c-Src, phosphatidylinositol 3'-kinase, and proteinkinase C activities and was dependent on the presence ofextracellular Ca²⁺. These results support the hypothesisthat the P2X₇ receptor and its signaling pathways play arole in astrocyte-mediated inflammation and neurodegenerative disease.

     

N-methyl-D-glucamine and propidium dyes utilize different permeation pathways at rat P2X(7) receptors

Activation of membrane P2X₇ receptors by extracellular ATP [or its analog 2',3'-O-(4-benzoylbenzoyl)-ATP] results in the opening within several milliseconds of an integral ion channel that is permeable to small cations. If the ATP application is maintained for several seconds, two further sequelae occur: there is a gradual increase in permeability to the larger cation N-methyl-D-glucamine and the cationic propidium dye quinolinium, 4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(triethylammonio)propyl]diiodide (YO-PRO-1) enters the cell. The similarity in the time course of these two events has led to the widespread view that N-methyl-D-glucamine and YO-PRO-1 enter through a common permeation pathway, the "dilating" P2X₇ receptor pore. Here we provide two independent lines of evidence against this view. We studied single human embryonic kidney cells expressing rat P2X₇ receptors with patch-clamp recordings of membrane current and with fluorescence measurements of YO-PRO-1 uptake. First, we found that maintained application of the ATP analog did not cause any increase in N-methyl-D-glucamine permeability when the extracellular solution contained its normal sodium concentration, although YO-PRO-1 uptake was readily observed. Second, we deleted a cysteine-rich 18-amino acid segment in the intracellular juxtamembrane region of the P2X₇ receptor. This mutated receptor showed normal YO-PRO-1 uptake but had no permeability to N-methyl-D-glucamine. Together, the clear differential effects of extracellular sodium ions or of mutation of the receptor strongly suggest that N-methyl-D-glucamine and YO-PRO-1 do not enter the cell by the same permeation pathway. ATP; cation channel; permeability; quinolinium, 4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(triethylammonio)propyl]diiodide     

Are second messengers crucial for opening the pore associated with P2X7 receptor?

Stimulation of the P2X₇ receptor by ATP induces cell membrane depolarization, increase in intracellular Ca²⁺ concentration, and, in most cases, permeabilization of the cell membrane to molecules up to 900 Da. After the activation of P2X₇, at least two phenomena occur: the opening of low-conductance (8 pS) cationic channels and pore formation. At least two conflicting hypotheses have been postulated to reconcile these findings: 1) the P2X₇ pore is formed as a result of gradual permeability increase (dilation) of cationic channels, and 2) the P2X₇ pore represents a distinct channel, possibly activated by a second messenger and not directly by extracellular nucleotides. In this study, we investigated whether second messengers are necessary to open the pore associated with the P2X₇ receptor in cells that expressed the pore activity by using the patch-clamp technique in whole cell and cell-attached configurations in conjunction with fluorescent imaging. In peritoneal macrophages and 2BH4 cells, we detected permeabilization and single-channel currents in the cell-attached configuration when ATP was applied outside the membrane patch in a condition in which oxidized ATP and Lucifer yellow were maintained within the pipette. Our data support Ca²⁺ as a second messenger associated with pore formation because the permeabilization depended on the presence of intracellular Ca²⁺ and was blocked by BAPTA-AM. In addition, MAPK inhibitors (SB-203580 and PD-98059) blocked the permeabilization and single-channel currents in these cells. Together our data indicate that the P2X₇ pore depends on second messengers such as Ca²⁺ and MAP kinases. electrophysiology; pore formation     

ATP stimulates Na+-glucose cotransporter activity via cAMP and p38 MAPK in renal proximal tubule cells

Extracellular ATP plays an important role in the regulation of renal function. However, the effect of ATP on the Na⁺-glucose cotransporters (SGLTs) has not been elucidated in proximal tubule cells (PTCs). Therefore, this study was performed to examine the action of ATP on SGLTs and their related signal pathways in primary cultured rabbit renal PTCs. ATP increased [¹⁴C]--methyl-D-glucopyranoside (-MG) uptake in a time-dependent (>1 h) and dose-dependent (>10^–6 M) manner. ATP stimulated -MG uptake by increasing in V_max without affecting K_m. ATP-induced increase of -MG uptake was correlated with the increase in both SGLT1 and SGLT2 protein expression levels. ATP-induced stimulation of -MG uptake was blocked by suramin (nonspecific P2 receptor antagonist), RB-2 (P2Y receptor antagonist), and MRS-2179 (P2Y₁ receptor antagonist), suggesting a role for the P2Y receptor. ATP-induced stimulation of -MG uptake was blocked by pertussis toxin (PTX, a G_i protein inhibitor), SQ-22536 (an adenylate cyclase inhibitor), and PKA inhibitor amide 14-22 (PKI). ATP also increased cAMP formation, which was blocked by PTX and RB-2. However, pretreatment of adenosine deaminase did not block ATP-induced cAMP formation. In addition, ATP-induced stimulation of -MG uptake was blocked by SB-203580 (p38 MAPK inhibitor), but not by PD-98059 (p44/42 MAPK inhibitor) or SP-600125 (JNK inhibitor). Indeed, ATP induced phosphorylation of p38 MAPK. In conclusion, ATP increases -MG uptake via cAMP and p38 MAPK in renal PTCs. adenosine 5'-triphosphate; mitogen-activated protein kinase     

P2X7 receptor-mediated apoptosis of human cervical epithelial cells

Normal human ectocervical epithelial (hECE) cells undergo apoptosis in culture. Baseline apoptosis could be increased by shifting cells to serum-free medium and blocked by lowering extracellular calcium. Treatment with the ATPase apyrase attenuated baseline apoptosis, suggesting that extracellular ATP and purinergic mechanisms control the apoptosis. Treatment with ATP and the P2X₇ receptor analog 2'-3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP) increased apoptosis significantly, in a time- and dose-related manner. The threshold of ATP effect was 0.5 µM in hECE cells and 1 µM in CaSki cancer cells. The apoptotic effect of BzATP was additive in part to that of tumor necrosis factor (TNF)-, and it could be attenuated by lowering extracellular calcium and by treatment with the caspase-9 inhibitor Leu-Glu-His-Asp-O-methyl-fluoromethylketone (LEHD-FMK). Treatment with BzATP activated caspase-9, and, in contrast to TNF-, it had only a mild effect on caspase-8. Both BzATP and TNF- activated caspase-3, suggesting that BzATP activates predominantly the mitochondrial apoptotic pathway. Both hECE and CaSki cells secrete ATP into the extracellular fluid, and mean ATP activity in conditioned medium was 0.5 µM, which is in the range of values that suffice to activate the P2X₇ receptor. On the basis of these findings we propose a novel autocrine-paracrine mechanism of cervical cell apoptosis that operates by P2X₇ receptor control of cytosolic calcium and utilizes the mitochondrial apoptotic pathway. cervix; epithelium; ATP; 2'-3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate     

Partial agonists and antagonists reveal a second permeability state of human lymphocyte P2Z/P2X7 channel

Extracellular ATP is known to trigger apoptosis of thymocytesand lymphocytes through a P2Z receptor at which ATP is a partial agonist, giving only 70% of the maximum response of3'-O-(4-benzoyl)benzoyl-adenosine 5'-triphosphate (BzATP), a full agonist. This cytolytic receptor and its associated ion channel areCa²⁺ (andBa²⁺) selective but also passmolecules up to the size of ethidium cation (314 Da).RT-PCR showed identity between lymphocyte P2Z and thehP2X₇ gene recently cloned fromhuman monocytes. When human leukemic B lymphocytes were incubated withATP and¹³³Ba²⁺,an immediate influx of isotope occurred. It was augmented by 45% whenATP was added 10 min before isotope. Time-resolved flow cytometry wasused to examine kinetics of ethidium uptake in cells incubated withBzATP or the partial agonists ATP, 2-methylthioadenosine 5'-triphosphate, or adenosine5'-O-(3-thiotriphosphate).Maximally effective concentrations of BzATP (50 µM) induced immediateuptake of ethidium at a rate linear with time. In contrast, a delay was observed (30 s) before ethidium uptake commenced after addition ofmaximally effective ATP concentrations (500 µM) at 37°C, and thedelay was longer at 24°C. ATP addition 2-10 min beforeethidium abolished the delay. The delay was longer with other partialagonists and inversely related to maximal flux produced by agonist. Adelay was also observed for submaximal BzATP concentrations (10-20µM). P2Z/P2X₇ inhibitors, KN-62and5-(N,N-hexamethylene)-amiloride, reduced the rate of agonist-induced ethidium uptake and lengthened thedelay. The results support a model in which agonists forP2Z/P2X₇ receptor mediate animmediate channel opening allowing passage of small inorganic cations,followed by a slow further permeability increase allowing passage oflarger permeant cations like ethidium. The rate of the second stepdepends on time and temperature and the efficacy and concentration ofagonist and is slowed by antagonists, suggesting it depends on thefraction of P2Z/P2X₇ channels held in the initial openstate.