Inhibition of the human P2X7 receptor by a novel protein tyrosine kinase antagonist

A panel of 18 protein tyrosine kinase antagonists were tested for their inhibitory effect on human P2X₇ receptor-mediated ⁸⁶Rb⁺ (K⁺) efflux. The most potent compound (compound P), a phthalazinamine derivative and an inhibitor of vascular endothelial growth factor receptor kinase, blocked ATP-induced ⁸⁶Rb⁺-efflux in human B-lymphocytes and erythrocytes by 76% and 66%, respectively. This inhibition was dose-dependent in both cell types with an IC₅₀ of ∼5 μM. Kinetic analysis showed compound P was a non-competitive inhibitor of P2X₇. This compound also inhibited ATP-induced ethidium⁺ influx into B-lymphocytes and P2X₇-transfected-HEK-293 cells, as well as ATP-induced ⁸⁶Rb⁺-efflux from canine erythrocytes. Externally, but not internally, applied compound P impaired ATP-induced inward currents in P2X₇-transfected-HEK-293 cells. This study demonstrates that a novel protein tyrosine kinase antagonist directly impairs native and recombinant human P2X₇ receptors. The data suggests that antagonists which target ATP-binding sites of kinases may potentially block the P2X₇ receptor.     

TGF-β1 prevents up-regulation of the P2X7 receptor by IFN-γ and LPS in leukemic THP-1 monocytes

The P2X7 receptor is an extracellular ATP-gated cation channel critical in inflammation and immunity, and can be up-regulated by IFN-γ and LPS. This study aimed to examine the effect of TGF-β1 on the up-regulation of P2X7 function and expression in leukemic THP-1 monocytes differentiated with IFN-γ and LPS. Cell-surface molecules including P2X7 were examined by immunofluorescence staining. Total P2X7 protein and mRNA was assessed by immunoblotting and RT-PCR respectively. P2X7 function was evaluated by ATP-induced cation dye uptake measurements. Cell-surface P2X7 was present on THP-1 cells differentiated for 3 days with IFN-γ and LPS but not on undifferentiated THP-1 cells. ATP induced ethidium⁺ uptake into differentiated but not undifferentiated THP-1 cells, and the P2X7 antagonist, KN-62, impaired ATP-induced ethidium⁺ uptake. Co-incubation of cells with TGF-β1 plus IFN-γ and LPS prevented the up-regulation of P2X7 expression and ATP-induced ethidium⁺ uptake in a concentration-dependent fashion with a maximum effect at 5 ng/ml and with an IC₅₀ of ~ 0.4 ng/ml. Moreover, ATP-induced YO-PRO-1²⁺ uptake and IL-1β release were abrogated in cells co-incubated with TGF-β1. TGF-β1 also abrogated the amount of total P2X7 protein and mRNA induced by IFN-γ and LPS. Finally, TGF-β1 prevented the up-regulation of cell-surface CD86, but not CD14 and MHC class II, by IFN-γ and LPS. These results indicate that TGF-β1 prevents the up-regulation of P2X7 function and expression by IFN-γ and LPS in THP-1 monocytes. This suggests that TGF-β1 may limit P2X7-mediated processes in inflammation and immunity.     

P2X7 receptor activation induces cell death and CD23 shedding in human RPMI 8226 multiple myeloma cells

Background

The extracellular ATP-gated cation channel, P2X7 receptor, has an emerging role in neoplasia, however progress in the field is limited by a lack of malignant cell lines expressing this receptor.

Methods

Immunofluorescence labelling and a fixed-time ATP-induced ethidium⁺ uptake assay were used to screen a panel of human malignant cell lines for the presence of functional P2X7. The presence of P2X7 was confirmed by RT-PCR, immunoblotting and pharmacological approaches. ATP-induced cell death was measured by colourimetric tetrazolium-based and cytofluorometric assays. ATP-induced CD23 shedding was measured by immunofluorescence labelling and ELISA.

Results

RPMI 8226 multiple myeloma cells expressed P2X7 mRNA and protein, as well as P2X1, P2X4 and P2X5 mRNA. ATP induced ethidium⁺ uptake into these cells with an EC₅₀ of ~ 116 μM, and this uptake was reduced in the presence of extracellular Ca²⁺ and Mg²⁺. The P2X7 agonist 2'- and 3'-0(4-benzoylbenzoyl) ATP, but not UTP, induced ethidium⁺ uptake. ATP-induced ethidium⁺ uptake was impaired by the P2X7 antagonists, KN-62 and A-438079. ATP induced death and CD23 shedding in RPMI 8226 cells, and both processes were impaired by P2X7 antagonists. The metalloprotease antagonists, BB-94 and GM6001, impaired ATP-induced CD23 shedding but not ethidium⁺ uptake.

Conclusions

P2X7 receptor activation induces cell death and CD23 shedding in RPMI 8226 cells.

General significance

RPMI 8226 cells may be useful to study the role of P2X7 in multiple myeloma and B-lymphocytes.     

P2X7 receptor activation mediates organic cation uptake into human myeloid leukaemic KG-1 cells

The P2X7 purinergic receptor is an ATP-gated cation channel with an emerging role in neoplasia. In this study we demonstrate that the human KG-1 cell line, a model of acute myelogenous leukaemia, expresses functional P2X7. RT-PCR and immunochemical techniques demonstrated the presence of P2X7 mRNA and protein respectively in KG-l cells, as well as in positive control multiple myeloma RPMI 8226 cells. Flow cytometric measurements demonstrated that ATP induced ethidium⁺ uptake into KG-l cells suspended in sucrose medium (EC₅₀ of ∼3 μM), but not into cells in NaCl medium. In contrast, ATP induced ethidium⁺ uptake into RPMI 8226 cells suspended in either sucrose or NaCl medium (EC₅₀ of ∼3 or ∼99 μM, respectively), as well as into RPMI 8226 cells in KCl medium (EC₅₀ of ∼18 μM). BzATP and to a lesser extent ATPγS and αβ-methylene ATP, but not ADP or UTP, also induced ethidium⁺ uptake into KG-1 cells. ATP-induced ethidium⁺ uptake was completely impaired by the P2X7 antagonists, AZ10606120 and A-438079. ATP-induced ethidium⁺ uptake was also impaired by probenecid but not by carbenoxolone, both pannexin-1 antagonists. ATP induced YO-PRO-1²⁺ and propidium²⁺ uptake into KG-1 cells. Finally, sequencing of full-length P2X7 cDNA identified several single nucleotide polymorphisms (SNPs) in KG-1 cells including H155Y, A348T, T357S and Q460R. RPMI 8226 cells contained A348T, A433V and H521Q SNPs. In conclusion, the KG-1 cell line expresses functional P2X7. This cell line may help elucidate the signalling pathways involved in P2X7-induced survival and invasiveness of myeloid leukaemic cells.     

Probenecid directly impairs activation of the canine P2X7 receptor

The current study aimed to determine if probenecid could directly impair the canine P2X7 receptor, a ligand-gated cation channel activated by extracellular adenosine 5′-triphosphate (ATP). Patch clamp measurements demonstrated that probenecid impairs ATP-induced inward currents in HEK-293 cells expressing canine P2X7. Flow cytometric measurements of ethidium⁺ uptake into HEK-293 cells expressing canine P2X7 showed that probenecid impairs ATP-induced pore formation in a concentration-dependent manner, with a half maximal inhibitory concentration of 158 µM. Finally, ELISA measurements revealed that probenecid impairs ATP-induced interleukin-1β release in dog blood. In conclusion, this study reveals that probenecid can directly impair canine P2X7 activation.     

Canine erythrocytes express the P2X7 receptor: greatly increased function compared with human erythrocytes

Over three decades ago, Parker and Snow (Am J Physiol 223: 888-893, 1972) demonstrated that canine erythrocytes undergo an increase in cation permeability when incubated with extracellular ATP. In this study we examined the expression and function of the channel/pore-forming P2X(7) receptor on canine erythrocytes. P2X(7) receptors were detected on canine erythrocytes by immunocytochemistry and immunoblotting. Extracellular ATP induced (86)Rb(+) (K(+)) efflux from canine erythrocytes that was 20 times greater than that from human erythrocytes. The P2X(7) agonist 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-trisphosphate (BzATP) was more potent than ATP, and both stimulated (86)Rb(+) efflux from erythrocytes in a dose-dependent fashion with EC(50) values of approximately 7 and approximately 309 microM, respectively. 2-Methylthioadenosine 5'-triphosphate and adenosine 5'-O-(3-thiotriphosphate) induced a smaller (86)Rb(+) efflux from erythrocytes, whereas ADP, AMP, UTP, or adenosine had no effect. ATP-induced (86)Rb(+) efflux from erythrocytes was inhibited by oxidized ATP, KN-62, and Brilliant blue G, known P2X(7) antagonists. ATP also induced uptake of choline(+) into canine erythrocytes that was 60 times greater than that into human erythrocytes. Overnight incubation of canine erythrocytes with ATP and BzATP induced phosphatidylserine exposure in >80% of cells and caused up to 20% hemolysis. In contrast, <30% of human erythrocytes showed phosphatidylserine exposure after overnight incubation with ATP and BzATP, and hemolysis was negligible. Flow cytometric measurements of ATP-induced ethidium(+) uptake showed that P2X(7) function was three times lower in canine monocytes than in human monocytes. These data show that the massive cation permeability increase induced by extracellular ATP in canine erythrocytes results from activation and opening of the P2X(7) receptor channel/pore.     

P2X(7) receptor activation causes phosphatidylserine exposure in human erythrocytes

Activation of cation channels causes erythrocyte phosphatidylserine (PS) exposure and cell shrinkage. Human erythrocytes express the P2X₇ receptor, an ATP-gated cation channel. The two most potent P2X₇ agonists, BzATP and ATP, stimulated PS exposure in human erythrocytes. Other nucleotides also induced erythrocyte PS exposure with an order of agonist potency of BzATP > ATP > 2MeSATP > ATPγS; however neither ADP nor UTP had an effect. ATP induced PS exposure in erythrocytes in a dose-dependent fashion with an EC₅₀ of ∼75 μM. BzATP- and ATP-induced erythrocyte PS exposure was impaired by oxidised ATP, as well as in erythrocytes from subjects who had inherited loss-of-function polymorphisms in the P2X₇ receptor. ATP-induced PS exposure in erythrocytes was not significantly altered in the presence of EGTA excluding a role for extracellular Ca²⁺. These results show that P2X₇ activation by extracellular ATP can induce PS exposure in erythrocytes.     

P2X7 receptor activation induces cell death and microparticle release in murine erythroleukemia cells

Extracellular ATP induces cation fluxes in and impairs the growth of murine erythroleukemia (MEL) cells in a manner characteristic of the purinergic P2X7 receptor, however the presence of P2X7 in these cells is unknown. This study investigated whether MEL cells express functional P2X7. RT-PCR, immunoblotting and immunofluorescence staining demonstrated the presence of P2X7 in MEL cells. Cytofluorometric measurements demonstrated that ATP induced ethidium⁺ uptake into MEL cells in a concentration-dependent fashion and with an EC₅₀ of ∼ 154 μM. The most potent P2X7 agonist 2′- and 3′-0(4-benzoylbenzoyl) ATP, but not ADP or UTP, induced ethidium⁺ uptake. ATP-induced ethidium⁺ and YO-PRO-1²⁺ uptake were impaired by the P2X7 antagonist, A-438079. A colourmetric assay demonstrated that ATP impaired MEL cell growth. A cytofluorometric assay showed that ATP induced MEL cell death and that this process was impaired by A-438079. Finally, cytofluorometric measurements of Annexin-V binding and bio-maleimide staining demonstrated that ATP could induce rapid phosphatidylserine exposure and microparticle release in MEL cells respectively, both of which were impaired by A-438079. These results demonstrate that MEL cells express functional P2X7, and indicate that activation of this receptor may be important in the death and release of microparticles from red blood cells in vivo.     

Pannexin1 channels act downstream of P2X7 receptors in ATP-induced murine T-cell death

Death of murine T cells induced by extracellular ATP is mainly triggered by activation of purinergic P2X₇ receptors (P2X₇Rs). However, a link between P2X₇Rs and pannexin1 (Panx1) channels, which are non-selective, has been recently demonstrated in other cell types. In this work, we characterized the expression and cellular distribution of pannexin family members (Panxs 1, 2 and 3) in isolated T cells. Panx1 was the main pannexin family member clearly detected in both helper (CD4⁺) and cytotoxic (CD8⁺) T cells, whereas low levels of Panx2 were found in both T-cell subsets. Using pharmacological and genetic approaches, Panx1 channels were found to mediate most ATP-induced ethidium uptake since this was drastically reduced by Panx1 channel blockers (¹⁰Panx1, Probenecid and low carbenoxolone concentration) and absent in T cells derived from Panx1^?/? mice. Moreover, electrophysiological measurements in wild-type CD4⁺ cells treated with ATP unitary current events and pharmacological sensitivity compatible with Panx1 channels were found. In addition, ATP release from T cells treated with 4Br-A23187, a calcium ionophore, was completely blocked with inhibitors of both connexin hemichannels and Panx1 channels. Panx1 channel blockers drastically reduced the ATP-induced T-cell mortality, indicating that Panx1 channels mediate the ATP-induced T-cell death. However, mortality was not reduced in T cells of Panx1^?/? mice, in which levels of P2X₇Rs and ATP-induced intracellular free Ca²⁺ responses were enhanced suggesting that P2X₇Rs take over Panx1 channels lose-function in mediating the onset of cell death induced by extracellular ATP.     

CAY10593 inhibits the human P2X7 receptor independently of phospholipase D1 stimulation

The P2X7 receptor is a trimeric ATP-gated cation channel important in health and disease. We have observed that the specific phospholipase D (PLD)1 antagonist, CAY10593 impairs P2X7-induced shedding of the ‘low affinity’ IgE receptor, CD23. The current study investigated the mode of action of this compound on P2X7 activation. Measurements of ATP-induced ethidium⁺ uptake revealed that CAY10593 impaired P2X7-induced pore formation in human RPMI 8226 B cells, P2X7-transfected HEK-293 cells and peripheral blood mononuclear cells. Concentration response curves demonstrated that CAY10593 impaired P2X7-induced pore formation in RPMI 8226 cells more potently than the PLD2 antagonist CAY10594 and the non-specific PLD antagonist halopemide. Electrophysiology measurements demonstrated that CAY10593 also inhibited P2X7-induced inward currents. Notably, RT-PCR demonstrated that PLD1 was absent in RPMI 8226 cells, while choline-Cl medium or 1-butanol, which block PLD stimulation and signalling respectively did not impair P2X7 activation in these cells. This data indicates that CAY10593 impairs human P2X7 independently of PLD1 stimulation and highlights the importance of ensuring that compounds used in signalling studies downstream of P2X7 activation do not affect the receptor itself.     

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.

     

Pannexin1 channels act downstream of P2X7 receptors in ATP-induced murine T-cell death

Death of murine T cells induced by extracellular ATP is mainly triggered by activation of purinergic P2X₇ receptors (P2X₇Rs). However, a link between P2X₇Rs and pannexin1 (Panx1) channels, which are non-selective, has been recently demonstrated in other cell types. In this work, we characterized the expression and cellular distribution of pannexin family members (Panxs 1, 2 and 3) in isolated T cells. Panx1 was the main pannexin family member clearly detected in both helper (CD4⁺) and cytotoxic (CD8⁺) T cells, whereas low levels of Panx2 were found in both T-cell subsets. Using pharmacological and genetic approaches, Panx1 channels were found to mediate most ATP-induced ethidium uptake since this was drastically reduced by Panx1 channel blockers (¹⁰Panx1, Probenecid and low carbenoxolone concentration) and absent in T cells derived from Panx1^−/− mice. Moreover, electrophysiological measurements in wild-type CD4⁺ cells treated with ATP unitary current events and pharmacological sensitivity compatible with Panx1 channels were found. In addition, ATP release from T cells treated with 4Br-{"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187, a calcium ionophore, was completely blocked with inhibitors of both connexin hemichannels and Panx1 channels. Panx1 channel blockers drastically reduced the ATP-induced T-cell mortality, indicating that Panx1 channels mediate the ATP-induced T-cell death. However, mortality was not reduced in T cells of Panx1^−/− mice, in which levels of P2X₇Rs and ATP-induced intracellular free Ca²⁺ responses were enhanced suggesting that P2X₇Rs take over Panx1 channels lose-function in mediating the onset of cell death induced by extracellular ATP.     

Paroxetine suppresses recombinant human P2X7 responses

P2X7 receptor (P2X7) activity may link inflammation to depressive disorders. Genetic variants of human P2X7 have been linked with major depression and bipolar disorders, and the P2X7 knockout mouse has been shown to exhibit anti-depressive-like behaviour. P2X7 is an ATP-gated ion channel and is a major regulator of the pro-inflammatory cytokine interleukin 1β (IL-1β) secretion from monocytes and microglia. We hypothesised that antidepressants may elicit their mood enhancing effects in part via modulating P2X7 activity and reducing inflammatory responses. In this study, we determined whether common psychoactive drugs could affect recombinant and native human P2X7 responses in vitro. Common antidepressants demonstrated opposing effects on human P2X7-mediated responses; paroxetine inhibited while fluoxetine and clomipramine mildly potentiated ATP-induced dye uptake in HEK-293 cells stably expressing recombinant human P2X7. Paroxetine inhibited dye uptake mediated by human P2X7 in a concentration-dependent manner with an IC₅₀ of 24 μM and significantly reduces ATP-induced inward currents. We confirmed that trifluoperazine hydrochloride suppressed human P2X7 responses (IC₅₀ of 6.4 μM). Both paroxetine and trifluoperazine did not inhibit rodent P2X7 responses, and mutation of a known residue (F 95L) did not alter the effect of either drug, suggesting neither drug binds at this site. Finally, we demonstrate that P2X7-induced IL-1β secretion from lipopolysaccharide (LPS)-primed human CD14⁺ monocytes was suppressed with trifluoperazine and paroxetine.     

Involvement of P2X receptors in the NAD+-induced rise in [Ca2+]i in human monocytes

In the present study, we show that the extracellular addition of nicotinamide adenine dinucleotide (NAD⁺) induces a transient rise in [Ca²⁺]_i in human monocytes caused by an influx of extracellular calcium. The NAD⁺-induced Ca²⁺ response was prevented by adenosine triphosphate (ATP), suggesting the involvement of ATP receptors. Of the two subtypes of ATP receptors (P2X and P2Y), the P2X receptors were considered the most likely candidates. By the use of subtype preferential agonists and antagonists, we identified P2X₁, P2X₄, and P2X₇ receptors being engaged in the NAD⁺-induced rise in [Ca²⁺]_i. Among the P2X receptor subtypes, the P2X₇ receptor is unique in facilitating the induction of nonselective pores that allow entry of ethidium upon stimulation with ATP. In monocytes, opening of P2X₇ receptor-dependent pores strongly depends on specific ionic conditions. Measuring pore formation in response to NAD⁺, we found that NAD⁺ unlike ATP lacks the ability to induce this pore-forming response. Whereas as little as 100 μM ATP was sufficient to activate the nonselective pore, NAD⁺ at concentrations up to 2 mM had no effect. Taken together, these data indicate that despite similarities in the action of extracellular NAD⁺ and ATP there are nucleotide-specific variations. So far, common and distinct features of the two nucleotides are only beginning to be understood.     

Direct Observation of ATP-Induced Conformational Changes in Single P2X4 Receptors

The ATP-gated P2X₄ receptor is a cation channel, which is important in various pathophysiological events. The architecture of the P2X₄ receptor in the activated state and how to change its structure in response to ATP binding are not fully understood. Here, we analyze the architecture and ATP-induced structural changes in P2X₄ receptors using fast-scanning atomic force microscopy (AFM). AFM images of the membrane-dissociated and membrane-inserted forms of P2X₄ receptors and a functional analysis revealed that P2X₄ receptors have an upward orientation on mica but lean to one side. Time-lapse imaging of the ATP-induced structural changes in P2X₄ receptors revealed two different forms of activated structures under 0 Ca²⁺ conditions, namely a trimer structure and a pore dilation-like tripartite structure. A dye uptake measurement demonstrated that ATP-activated P2X₄ receptors display pore dilation in the absence of Ca²⁺. With Ca²⁺, the P2X₄ receptors exhibited only a disengaged trimer and no dye uptake was observed. Thus our data provide a new insight into ATP-induced structural changes in P2X₄ receptors that correlate with pore dynamics.     

Human neutrophils do not express purinergic P2X7 receptors

It has been reported that in human neutrophils, external ATP activates plasma membrane purinergic P2X₇ receptors (P2X₇R) to elicit Ca²⁺ entry, production of reactive oxygen species (ROS), processing and release of pro-inflammatory cytokines, shedding of adhesion molecules and uptake of large molecules. However, the expression of P2X₇R at the plasma membrane of neutrophils has also been questioned since these putative responses are not always reproduced. In this work, we used electrophysiological recordings to measure functional responses associated with the activation of membrane receptors, spectrofluorometric measurements of ROS production and ethidium bromide uptake to asses coupling of P2X₇R activation to downstream effectors, immune-labelling of P2X₇R using a fluorescein isothiocyanate-conjugated antibody to detect the receptors at the plasma membrane, RT-PCR to determine mRNA expression of P2X₇R and Western blot to determine protein expression in neutrophils and HL-60 cells. None of these assays reported the presence of P2X₇R in the plasma membrane of neutrophils and non-differentiated or differentiated HL-60 cells—a model cell for human neutrophils. We concluded that P2X₇R are not present at plasma membrane of human neutrophils and that the putative physiological responses triggered by external ATP should be reconsidered.     

Synthesis and structure–activity relationships of pyrazolodiazepine derivatives as human P2X7 receptor antagonists

Screening of library compounds has yielded pyrazolodiazepine derivatives with P2X₇ receptor antagonist activity. To explore the structure–activity relationships (SAR) of these pyrazolodiazepines as human P2X₇ receptor antagonists, derivatives were synthesized by substitutions at positions R² and R³ of the pyrazolodiazepine skeleton. Using a 2′(3′)-O-(4-benzoylbenzoyl)ATP (BzATP)-induced fluorescent ethidium uptake assay, the activities of these derivatives were tested in HEK-293 cells stably expressing human P2X₇ receptors. Moreover, the effect of these derivatives was assessed by measuring their effect on IL-1β release induced by BzATP-induced activation of differentiated THP-1 cells. A 2-phenethyl pyrazolodiazepine derivative with a 1-methyl-1H-3-indolyl group at position R² had fivefold greater activity than the derivative with a 5-isoquinolinyl at R². Moreover, a benzyl moiety at R³ had fivefold greater activity than a bicyclic moiety. The stereochemical effect at C-6 showed a preference for the (R)-isomer. Among the series of active derivatives, compound 23b, with a phenethyl group at R¹, a 3-methyl indole at R², and a benzyl at R³, exhibited activity similar to that of the positive control, KN-62, as shown by the inhibitory effects of IL-1β release.     

Expression and function of P2X(7) receptor and CD39/Entpd1 in patients with type 2 diabetes and their association with biochemical parameters

Chronic inflammation is an important contributor to the insulin resistance observed in type 2 diabetes (T2D). We evaluated the expression and function of the P2X₇ receptor and CD39/Entpd1, molecules involved in the cellular regulation of inflammation, in peripheral blood mononuclear cells from T2D patients, and their correlation with the concentration of HbA1c in blood. T2D patients with deficient metabolic control (DC) showed increased proportion of P2X₇⁺ cells compared with healthy individuals; T2D-DC subjects also displayed higher proportion of CD14⁺, CD4⁺ and CD19⁺ subpopulations of P2X₇⁺ cells when compared with T2D patients with acceptable metabolic control. A significant association was observed between the proportion of P2X₇⁺CD14⁺ cells and blood concentration of LDL-c. In addition, the percentages of CD39⁺ cells and CD39⁺CD19⁺ cells were significantly associated with HbA1c and fasting plasma glucose levels. No changes were observed in the function of P2X₇⁺ cells from T2D patients; however, enhanced CD39/Entpd1 enzyme activity and low serum levels of IL-17 were detected. Therefore, CD39⁺ cells could have a balancing regulatory role in the inflammatory process observed in patients with T2D.     

Connexin43 and pannexin1 channels in osteoblasts: who is the "hemichannel"?

Osteoblasts sense and respond to mechanical stimuli in a process involving influx and release of large ions and signaling molecules. Unapposed gap junction hemichannels formed of connexin43 (Cx43) have been proposed as a major route for such exchange, in particular for release of ATP and prostaglandin E₂ (PGE₂) in osteocytes. However, we have found that Cx43-null osteoblasts have unaltered, mechanically induced PGE₂ release and ATP-induced YoPro dye uptake. In contrast, PGE₂ release in response to fluid shear stress is abolished in P2X₇ receptor (P2X₇R)–null osteoblasts, and ATP-induced dye uptake is attenuated following treatment of wild-type cells with a P2X₇R or Pannexin1 (Panx1) channel blocker. These data indicate that Panx1 channels, in concert with P2X₇R, likely form a molecular complex that performs the hemichannel function in osteoblast mechanosignaling.     

Furosemide-sensitive K+ (Rb+) transport in human erythrocytes: Modes of operation,dependence on extracellular and intracellular Na+, kinetics,pH dependency and the effect of cell volume and N-ethylmaleimide

Summary The effect of extracellular and intracellular Na⁺ (Na _o ⁺ , Na _i ⁺ ) on ouabain-resistant, furosemide-sensitive (FS) Rb⁺ transport was studied in human erythrocytes under varying experimental conditions. The results obtained are consistent with the view that a (1 Na⁺+1 K⁺+2 Cl^–) cotransport system operates in two different modes: modei) promoting bidirectional 11 (Na⁺–K⁺) cotransport, and modeii) a Na _o ⁺ -independent 11 K _o ⁺ /K _i ⁺ exchange requiring Na _i ⁺ which, however, is not extruded. The activities of the two modes of operation vary strictly in parallel to each other among erythrocytes of different donors and in cell fractions of individual donors separated according to density. Rb⁺ uptake through Rb _o ⁺ /K _i ⁺ exchange contributes about 25% to total Rb⁺ uptake in 145mm NaCl media containing 5mm RbCl at normal Na _i ⁺ (pH 7.4). Na⁺–K⁺ cotransport into the cells occurs largely additive to K⁺/K⁺ exchange. Inward Na⁺–Rb⁺ cotransport exhibits a substrate inhibition at high Rb _o ⁺ . With increasing pH, the maximum rate of cotransport is accelerated at the expense of K⁺/K⁺ exchange (apparent pK close to pH 7.4). The apparentK _m ^Rb _o ⁺ of Na⁺–K⁺ cotransport is low (2mm) and almost independent of pH, and high for K⁺/K⁺ exchange (10 to 15mm), the affinity increasing with pH. The two modes are discussed in terms of a partial reaction scheme of (1 Na⁺+1 K⁺+2 Cl^–) cotransport with ordered binding and debinding, exhibiting a glide symmetry (first on outside = first off inside) as proposed by McManus for duck erythrocytes (McManus, T.J., 1987,Fed. Proc., in press). N-ethylmaleimide (NEM) chemically induces a Cl^–-dependent K⁺ transport pathway that is independent of both Na _o ⁺ and Na _i ⁺ . This pathway differs in many properties from the basal, Na _o ⁺ -independent K⁺/K⁺ exchange active in untreated human erythrocytes at normal cell volume. Cell swelling accelerates a Na _o ⁺ -independent FS K⁺ transport pathway which most probably is not identical to basal K⁺/K⁺ exchange. K _o ⁺ o ⁺

o ⁺ o ²⁺ reduce furosemide-resistant Rb⁺ inward leakage relative to choline _o ⁺ .