Effects Of Extracellular Adenosine Triphosphate Research Articles

Synergistic effect of extracellular adenosine triphosphate and quercetin on post-thaw quality and fertilization potential of Lohi ram sperm

This study determined the individual and combined effects of extracellular adenosine triphosphate (ATP) and quercetin (QUE) on the quality of post-thawed sperm and the fertilization potential of Lohi rams. In experiment 1, semen samples from four Lohi rams were pooled and extended with different concentrations of ATP or QUE (control; no ATP or QUE, 1 or 2 mM ATP and 10 or 20 μM QUE). In experiment 2, pooled semen samples were extended with various combinations of ATP and QUE (control; no ATP and QUE, 1 mM ATP + 10 μM QUE, 1 mM ATP + 20 μM QUE, 2 mM ATP + 10 μM QUE and 2 mM ATP + 20 μM QUE). All samples in both experiments were cryopreserved and analyzed for post-thawed sperm quality. In experiment 3, the best combination of ATP and QUE from experiment 2 was to extend semen, which was then used for laparoscopic insemination in estrus-synchronized ewes (n = 83). The results of experiment 1 showed that 1 mM ATP and 20 μM QUE treatments resulted in higher total motility, progressive motility, viability, plasma membrane intactness (PMI), and motion kinetics (VCL, VSL, VAP, LIN, and STR) compared to other treatments (p < 0.05). In experiment 2, the 1 mM ATP +10 μM QUE-treated group exhibited significantly higher total and progressive motility, PMI, and motion kinetics (VSL, VCL, VAP, STR, and BCF) compared to the control group (p < 0.05). In experiment 3, the fertilizing potential of sperms treated with 1 mM ATP +10 μM QUE was greater than that of untreated controls (58.1% vs. 27.5%, respectively, p-value = 0.012). In conclusion, the quality of post-thawed ram semen is enhanced when the extender is supplemented with extracellular 1 mM ATP and 20 μM QUE, whether used separately or in combination with 1 mM ATP and 10 μM QUE. Furthermore, the inclusion of 1 mM ATP and 10 μM QUE together in the extender significantly improves in vivo fertility in Lohi ram.

P2X7 receptor as the regulator of T-cell function in intestinal barrier disruption

The intestinal mucosa is a highly compartmentalized structure that forms a direct barrier between the host intestine and the environment, and its dysfunction could result in a serious disease. As T cells, which are important components of the mucosal immune system, interact with gut microbiota and maintain intestinal homeostasis, they may be involved in the process of intestinal barrier dysfunction. P2X7 receptor (P2X7R), a member of the P2X receptors family, mediates the effects of extracellular adenosine triphosphate and is expressed by most innate or adaptive immune cells, including T cells. Current evidence has demonstrated that P2X7R is involved in inflammation and mediates the survival and differentiation of T lymphocytes, indicating its potential role in the regulation of T cell function. In this review, we summarize the available research about the regulatory role and mechanism of P2X7R on the intestinal mucosa-derived T cells in the setting of intestinal barrier dysfunction.

Extracellular ATPs produced in seminal plasma exosomes regulate boar sperm motility and mitochondrial metabolism

Artificial insemination using diluted semen with reduced presence of seminal plasma has been applied worldwide. Sperm stored in seminal plasma rather than diluted or removed show improvement in survival and motility. However, the link between seminal plasma and sperm remains poorly understood. This study focuses on the effect of extracellular adenosine triphosphate (exATP) produced by boar seminal plasma exosomes on sperm motility, as well as the underlying molecular mechanisms. The seminal plasma exosomes had an average diameter of 86.6 nm and showed universal exosome markers, such as heat shock protein 70 (HSP70) and CD63. Production of net ATP increased when exosomes were incubated with glucose and partly inhibited by a glycolytic inhibitor such as iodoacetate. Fresh boar sperm incubated with exATP significantly increased sperm motility and reduced apoptotic rate. Ser21 phosphorylation of glycogen synthase kinase 3α (inactivation) also significantly increased, consistent with the increase in mitochondrial transmembrane potential in the exATP-treated sperm. Moreover, exATP treatment increased the intracellular ATP (inATP) concentration and decreased the ADP/ATP ratio in boar sperm. Lactate content in the incubation medium was decreased, whereas lactate dehydrogenase activity in sperm was increased. This finding suggested that exATP could prompt lactate to produce inATP in order to sustain motility. The combined results indicate that exATP produced in seminal plasma exosomes may finely modulate mitochondrial metabolism to control sperm motility. The results can provide insights into semen dilution and artificial insemination.

Multifaceted Effects of Extracellular Adenosine Triphosphate and Adenosine in the Tumor-Host Interaction and Therapeutic Perspectives.

Cancer is still one of the world’s most pressing health-care challenges, leading to a high number of deaths worldwide. Immunotherapy is a new developing therapy that boosts patient’s immune system to fight cancer by modifying tumor–immune cells interaction in the tumor microenvironment (TME). Extracellular adenosine triphosphate (eATP) and adenosine (Ado) are signaling molecules released in the TME that act as modulators of both immune and tumor cell responses. Extracellular adenosine triphosphate and Ado activate purinergic type 2 (P2) and type 1 (P1) receptors, respectively, triggering the so-called purinergic signaling. The concentration of eATP and Ado within the TME is tightly controlled by several cell-surface ectonucleotidases, such as CD39 and CD73, the major ecto-enzymes expressed in cancer cells, immune cells, stromal cells, and vasculature, being CD73 also expressed on tumor-associated fibroblasts. Once accumulated in the TME, eATP boosts antitumor immune response, while Ado attenuates or suppresses immunity against the tumor. In addition, both molecules can mediate growth stimulation or inhibition of the tumor, depending on the specific receptor activated. Therefore, purinergic signaling is able to modulate both tumor and immune cells behavior and, consequently, the tumor–host interaction and disease progression. In this review, we discuss the role of purinergic signaling in the host–tumor interaction detailing the multifaceted effects of eATP and Ado in the inflammatory TME. Moreover, we present recent findings into the application of purinergic-targeting therapy as a potential novel option to boost antitumor immune responses in cancer.

Proliferation of mouse liver stem/progenitor cells induced by plasma from patients with acute liver failure is modulated by P2Y2 receptor-mediated JNK activation

We recently reported that acute liver failure plasma (ALF-P) promotes the proliferation of mouse liver oval cells (OCs) through c-jun N-terminal kinase (JNK) activation. The aim of this study was to investigate the mechanism by which ALF-P induces JNK activation and OC proliferation. OCs and primary hepatocytes were exposed to ALF-P or normal control plasma (NC-P). Cell proliferation and activation of JNK and other JNK signaling molecules were detected subsequently. Next, we determined the effects of extracellular adenosine triphosphate (ATP) and ATP receptors on ALF-P-stimulated cell growth. Finally, the relationship between the tumor necrosis factor alpha (TNFα) and ATP receptor pathways was investigated. Cell proliferation accompanied by JNK activation was only observed in ALF-P-stimulated OCs. ALF-P stimulated the activation of SEK1/MKK4 and ATF2, but not c-Jun. Both PPADS (pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid) treatment and P2Y2 (G-protein-coupled) small interfering RNA (siRNA) transfection blocked the effects of ALF-P on cell proliferation and JNK activation. However, ATP levels in ALF-P were significantly lower than that in NC-P, and ATP did not stimulate the proliferation of OCs. On the other hand, TNFα stimulated JNK activation and proliferation of OCs. TNFα receptor antagonist partly inhibited the ALF-P-stimulated proliferation of OCs. Moreover, PPADS significantly inhibited TNFα-stimulated cell proliferation, induced apoptosis, and inhibited the activation of JNK. However, our data showed no significant difference in plasma TNFα levels between the NC-P and ALF-P samples. JNK activation induced by P2Y2 receptor crosstalk with the TNFα signaling pathway is important in mediating the effects of ALF-P on the proliferation and survival of OCs.

The effect of extracellular adenosine triphosphate on the spontaneous contractility of human myometrial strips

Objectives Extracellular ATP is involved in cell–cell signalling in a variety of tissues but its effects in tissue level signalling in the myometrium have been poorly studied to date. We hypothesised that extracellular ATP was involved in the control of myometrial contractile frequency and/or force. Study design In vitro study of the effect of altering the concentration of extracellular ATP on the spontaneous contractility of human myometrial strips obtained from term elective caesarean sections. Results Decreasing extracellular ATP levels by the ectoATP-ase agent apyrase VI (1–50 units/mL) produced a dose dependent decrease in contractile frequency (decrease of 46.3% compared with the baseline frequency at 20 units/mL, p = 0.001, n = 6). Contractility was unchanged by apyrase VII (20 units/mL), an agent with relatively greater ADPase activity, indicating an effect via ATP and not ADP. Contractile frequency increased after addition of ATP 10–100 μM (an increase to 145.8% of baseline frequency at 100 μM: 126.1–165.5%, p = 0.005, n = 7) or the ATPase inhibitor ARL at 100 μM (an increase to 136.3% of the baseline frequency: 107.1–165.5, p = 0.03, n = 7). Contractile force remained unchanged by these agents. Conclusions Extracellular ATP shows a dose–response relationship to contractile frequency but does not affect contractile force. Consequently it may be involved in the pacemaking mechanism for the generation of uterine contractions.

ATP elicits inward currents in isolated vasopressinergic neurohypophysial terminals via P2X2 and P2X3 receptors

Effects of extracellular adenosine tri-phosphate (ATP) on ionic currents were investigated using the perforated-patch whole-cell recording technique on isolated terminals of the Hypothalamic Neurohypophysial System (HNS). ATP induced a current response in 70% of these isolated terminals. This inwardly-rectifying, inactivating current had an apparent reversal near 0 mV and was dose-dependent on ATP with an EC50=9.6+/-1.0 microM. In addition, current amplitudes measured at maximal ATP concentrations and optimum holding potentials had a current density of 70.8 pA pF(-1) and were greatly inhibited by suramin and PPADS. Different purinergic receptor agonists were tested, with the following efficacy: ATP > or = 2-methylthioATP > ATP-gamma-S > Bz-Bz-ATP > alpha,beta-methylene-ATP > beta,gamma-methylene-ATP. However, UTP and ADP were ineffective. These data suggest the involvement of a P2X purinergic receptor in the ATP-induced responses. Immunocytochemical labeling in vasopressinergic terminals indicates the existence of P2X(2,3,4, and 7), but not P2X6 receptors. Additionally, P2X(2 and 3) were not found in terminals which labeled for oxytocin. In summary, the EC50, decay, inactivation, and pharmacology indicate that a functional mixture of P2X(2 and 3) homomeric receptors mediate the majority of the ATP responses in vasopressinergic HNS terminals. We speculate that the characteristics of these types of receptors reflect the function of co-released ATP in the terminal compartment of these and other CNS neurons.

Correlated ATP‐Induced Changes in Membrane Area and Membrane Conductance in Single Rat Adipocytes

In the past few years it has been shown that, like many other non-neuroendocrine cells, adipocytes possess a mechanism for triggered exocytosis. Endocytosis and exocytosis affect the plasma membrane surface area, which can be directly monitored with electrophysiological patch-clamp techniques by measuring membrane capacitance, a parameter linearly related to the plasma membrane area. In this study we used the whole-cell mode of the patch-clamp technique to measure changes in membrane capacitance to monitor the effect of extracellular adenosine triphosphate (ATP) on the dynamics of membrane area changes in single adipocytes. Experimental evidence shows that extracellular application of ATP (100 microM) increases membrane capacitance for 30 +/- 2%. In controls a significantly smaller increase of 3 +/- 2% was measured, which is due to a slow exocytic-endocytic membrane cycling rate of 0.3%/min. We found that ATP induces a transient increase in membrane current, temporally associated with the peak rate in membrane capacitance increase. These results show directly the presence of ATP-induced increase in membrane area correlated to the increase in membrane current in single adipocytes.

Inhibitory effects of extracellular adenosine triphosphate on growth of esophageal carcinoma cells

To study the growth inhibitory effects of ATP on TE-13 human squamous esophageal carcinoma cells in vitro. MTT assay was used to determine the inhibition of proliferation of ATP or adenosine (ADO) on TE-13 cell line. The morphological changes of TE-13 cells induced by ATP or ADO were observed under fluorescence light microscope by acridine orange (AO)/ethidium bromide (EB) double stained cells. The internucleosomal fragmentation of genomic DNA was detected by agarose gel electrophoresis. The apoptotic rate and cell cycle after treatment with ATP or ADO were determined by flow cytometry. ATP and ADO produced inhibitory effects on TE-13 cells at the concentration between 0.01 and 1.0 mmol/L. The IC(50) of TE-13 cells exposed to ATP or ADO for 48 and 72 h was 0.71 or 1.05, and 0.21 or 0.19 mmol/L, respectively. The distribution of cell cycle phase and proliferation index (PI) value of TE-13 cells changed, when being exposed to ATP or ADO at the concentrations of 0.01, 0.1, and 1 mmol/L for 48 h. ATP and ADO inhibited the cell proliferation by changing the distribution of cell cycle phase via either G(0)/G(1) phase (ATP or ADO, 1 mmol/L) or S phase (ATP, 0.1 mmol/L) arrest. Under light microscope, the tumor cells exposed to 0.3 mmol/L ATP or ADO displayed morphological changes of apoptosis. A ladder-like pattern of DNA fragmentation was obtained from TE-13 cells treated with 0.1-1 mmol/L ATP or ADO in agarose gel electrophoresis. ATP and ADO induced apoptosis of TE-13 cells in a dose-dependent manner at the concentration between 0.03 and 1 mmol/L. The maximum apoptotic rate of TE-13 cells exposed to ATP or ADO for 48 h was 16.63% or 16.9%, respectively. ATP and ADO inhibit cell proliferation, arrest cell cycle, and induce apoptosis of TE-13 cell line.

ATP-stimulated interleukin-6 synthesis through P2Y receptors on human osteoblasts

We investigated the effect of extracellular adenosine triphosphate (ATP) on the production of interleukin (IL)-6, whose molecules are capable of stimulating the development of osteoclasts from their hematopoietic precursors as well as are involved in signal transduction systems in human osteoblastic SaM-1 cells. These human osteoblasts constitutively expressed P2X4, P2X5, P2X6, P2Y2, P2Y5, and P2Y6 purinergic receptors. ATP increased gene- and protein-expression of IL-6 in SaM-1 cells. The expression of the IL-6 mRNA was maximal at 1 h, and the increase in IL-6 synthesis in response to ATP (10–100 μM) occurred in a concentration-dependent manner. Over the same concentration range of the nucleotide that was effective for IL-6 synthesis, ATP caused an increase in the intracellular Ca 2+ concentration ([Ca 2+] i), which increase was inhibited by pretreatment with suramin, a P2Y receptor antagonist, or 2-aminoethoxydiphenyl borate (2-APB), an inositol 1,4,5-trisphosphate receptor blocker, but not by the extracellular Ca 2+-chelating agent EGTA. The pretreatment of SaM-1 cells with suramin or 2-APB also inhibited the increase in IL-6 synthesis in response to ATP. These findings suggest that extracellular ATP-induced IL-6 synthesis occurs through P2Y receptors and mobilization of Ca 2+ from internal stores in human osteoblastic cells.

ATP activates nuclear translocation of mitogen-activated protein kinases in human granulosa-luteal cells

To investigate the specific role of adenosine triphosphate (ATP) in the subsequent MAPK-induced signaling cascade in human granulosa-luteal cells (hGLCs). We have previously demonstrated an effect of extracellular adenosine triphosphate (ATP) on ERKs and gonadotropin-induced progesterone secretion, implicating the significance of ATP in the regulation of ovarian function. The present study was designed to examine the effect of ATP on the activation of the MAPK signaling pathway, including nuclear translocation in hGLCs. Human GLCs were collected from patients undergoing in vitro fertilization programs. Human GLCs were cultured in FBS-supplemented DMEM for 3 days prior to our studies. Cells were starved in serum-free medium for 4h prior to subsequent treatments. To examine the dose-response relationship, hGLCs were treated with increasing concentrations of ATP (100nM, 1μ, 10μ, 100μ) for 5 min. For time-course experiments, hGLCs were treated with 10μM ATP for 1, 5, 10, 20 min. To determine the translocation of MAPKs, hGLCs were fixed 5 min after 10μM ATP exposure. Western blot analysis was performed using antibodies against the phosphorylated forms of ERK1 and ERK2 (P-MAPK, p42mapk and p44mapk, respectively), p42/p44 MAPK (total MAPK, T- MAPK), phospho JNK and activated P38. To examine the translocation of activated ERKs, FITC-conjugated secondary antibody was used to detect the distribution of total and phosphorylated ERKs. Western blot analysis, which detected the total and phosphorylated forms of ERK1 and ERK2, demonstrated that exogenous ATP evoked ERKs in a dose- and time-dependent manner. In contrast, p38 and JNK were not significantly activated following ATP treatment. Immunofluorescent staining revealed that phosphorylated ERKs were translocated from cytoplasm into nucleus subsequent to 10μ ATP treatment. To our knowledge, this is the first demonstration of the ATP-induced nuclear translocation of MAPKs in the human ovary. These results suggest that the MAPKs signaling pathway plays a role in mediating ATP actions in the human ovary.

P2U-receptor mediated endothelium-dependent but nitric oxide-independent vascular relaxation.

1. The dilator effect of extracellular adenosine triphosphate (ATP) has mainly been characterized as a direct effect on smooth muscle or as an endothelium-dependent effect mediated by nitric oxide (NO) or prostaglandins. We tested the hypothesis that endothelium-derived hyperpolarizing factor (EDHF) may also be involved. Dilator effects were studied in vitro by continuous recording of isomeric tension in cylindrical segments of rat blood vessels precontracted by noradrenaline (NA), in the presence of indomethacin (10 microM). 2. By screening different blood vessels in the rat we found that both acetylcholine (ACh) and ATP dilate mesenteric arteries with a resting tone of 1 mN by an endothelium-dependent non-NO mechanism. With an increased resting tone (4 mN) the dilatation was mediated by NO. Thus by varying the resting tension the different dilator mechanisms could be examined. However, in the carotid artery the dilatation was solely mediated by an endothelium-dependent NO mechanism, even at different resting tones (1 and 4 mN). 3. The N-nitro-L-arginine methyl ester (L-NAME)-resistant dilatation to ACh and ATP was further inhibited by the NO-scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), indicating L-NAME insensitive NO-synthesis. 4. In carotid arteries and mesenteric arteries at high resting tones (4 mN) the ATP-dilatation was totally inhibited by endothelium removal or L-NAME (10(-3) M). In mesenteric arteries at low resting tone (1 mN) the ATP, UTP (uridine-triphosphate) and 2-MeSATP (2methylthioATP)-dilatation was totally inhibited by endothelium removal. However, L-NAME in combination with indomethacin attenuated only 5% of the UTP dilatation, 70% of the ATP dilatation but all of the 2-MeSATP-dilatation. The inhibitors of Ca2+-activated K+ channels charybdotoxin (0.5 x 10(-7) M) together with apamin (10(-6) M), and the cytochrome P450 inhibitor, SKF 525A (10(-4) M), each in combination with indomethacin. L-NAME and PTIO (0.5 x 10(-3) M) totally abolished the remaining ATP and UTP-dilatation. This indicates a dilatation mediated by an endothelium-dependent non-NO factor, probably EDHF. 5. Agonist potency (UTP>ATP>>2-MeSATP), indicates that the EDHF-mediated dilatation was stimulated by a P2U-receptor, possibly by a selective pyrimidine-receptor. In contrast, a P2Y-receptor stimulated NO-mediated dilatation (2-MeSATP=ATP>UTP). 6. In conclusion, the dilator effects of ATP and especially UTP can be mediated by an endothelium-dependent non-NO-mediated mechanism, probably EDHF, mediated by a P2U-receptor, possibly a selective pyrimidine-receptor, while NO-mediated dilatation is stimulated mainly by a P2Y1-receptor. Furthermore, the EDHF-dilatation is dependent on the resting tone of the blood vessel.

Effects of extracellular ATP and adenosine on different thymocyte subsets: possible role of ATP-gated channels and G protein-coupled purinergic receptor.

To explore the possible role of purinergic receptors in thymocyte development and in pathogenesis of adenosine deaminase SCID, we studied effects of extracellular adenosine triphosphate (ATP(ext)) and adenosine on TCR- and steroid hormone-triggered processes in mouse thymocytes. Reverse transcriptase-PCR analysis confirms the mRNA expression of several types of purinergic receptors, while the functioning of ATP receptors in thymocytes is reflected by ATP(ext)-induced intracellular calcium increases and by thymocyte subset-specific sensitivity to the effects of ATP(ext) and adenosine. Only ATP(ext), but not the ATP catabolites, adenosine, dexamethasone, or TCR cross-linking, was efficient in triggering rapid protein synthesis independent lysis of CD4+8- thymocytes and peripheral CD4+ T cells. In contrast, extracellular adenosine specifically induced the apoptosis of CD4+8+ thymocytes. ATP(ext) also induced a slower process of DNA fragmentation and protein synthesis-dependent apoptosis in all thymocyte subsets. ATP(ext) had an additive effect with TCR cross-linking in the induction of thymocyte death, but, unexpectedly, the effects of ATP(ext) at high concentration were antagonistic to steroid-induced apoptosis. Described here, the properties of ATP(ext) and adenosine are consistent with their involvement in the regulation of T cell development due to differential expression and signaling through purinergic receptors in different thymocyte subsets. The possible role of purinergic receptor signaling in T cell differentiation and adenosine deaminase SCID is discussed.

Cytotoxic effect of extracellular ATP on L1210 leukemic cells and normal hemopoietic stem cells

The cytotoxic effect of extracellular adenosine triphosphate (ATP) was examined on normal murine hemopoietic stem cells and a representative leukemic cell line (L1210). After L1210 cells were incubated with 4 mM ATP for 3 h, 3H-thymidine incorporation was almost completely inhibited. The number of viable L1210 cells was also significantly decreased and L1210 colony formation was suppressed to approximately 30% of the control level after treatment. The CFU-GM survival rate was reduced to 70%, however, CFU-S and marrow nucleated cell numbers were not changed after the same treatment with ATP. All mice that were injected with the untreated mixture of normal marrow cells (3.3 × 10 4) and L1210 cells (3.3 × 10 3) died of leukemia within 18 days. On the contrary, 85% of the recipients given ATP-treated grafts survived more than 70 days. These findings indicate that ATP extra vivo treatment is useful for purging the residual leukemic cells in autologous bone marrow transplantation.

Modulation of L-type Ca2+ current by extracellular ATP in ferret isolated right ventricular myocytes.

1. The effects of extracellular adenosine triphosphate (ATP) on the basal L-type Ca2+ current (ICa) were investigated in ferret isolated right ventricular myocytes using the gigaohm seal voltage clamp in the whole-cell and cell-attached configurations. 2. Micromolar levels of extracellular ATP reversibly inhibited ICa in a concentration-dependent manner, without any significant changes in the voltage dependence of either the peak ICa I-V relationship or steady-state activation curve. 3. In contrast, micromolar levels of extracellular ATP did significantly alter the inactivation characteristics of ICa. Ten micromolar ATP: (i) increased the degree of steady-state inactivation of ICa; (ii) altered the time constants of ICa inactivation at 0 mV; and (iii) decreased the time constant of ICa recovery from inactivation at -70 mV. 4. The inhibitory effect of ATP on ICa was not blocked by atropine, a muscarinic cholinergic receptor antagonist, or CPDPX (8-cyclopentyl-3,4-dipropylxanthine), an A1 adenosine receptor antagonist. In contrast, the inhibitory effect of 10 microM ATP could be nearly completely antagonized by 100 microM suramin, a purinergic P2 receptor antagonist. 5. The potency order of ATP analogues in inhibiting ICa was 2-methyl-thio-ATP > ATP > alpha,beta-methylene-ATP, indicating involvement of a P2Y-type ATP receptor. 6. Pretreatment of cells with pertussis toxin (PTX) did not prevent the ATP-induced decrease in ICa. However, (i) ATP produced an irreversible decrease of ICa in the presence of intracellular GTP gamma S, and (ii) the inhibitory effect was significantly attenuated in the presence of intracellular GDP beta S, indicating the involvement of a PTX-insensitive G protein in the P2Y receptor-coupling process. 7. Neither (i) replacing extracellular Ca2+ with 1 mM Ba2+, nor (ii) intracellular perfusion of 10 mM BAPTA for at least 30 min attenuated the inhibitory effect of ATP on the current through Ca2+ channels, suggesting that the inhibitory effect was not obligatorily dependent upon influx of Ca2+ or changes in [Ca2+]i. 8. Ensemble-average current behaviour constructed from cell-attached patch recordings of single L-type Ca2+ channels (110 mM BaCl2) demonstrated that when 10 microM ATP was added to the superfusate on the outside of the patch electrode the inhibition of ICa was still observed, providing evidence for the involvement of intracellular diffusible second messenger(s).(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of extracellular adenosine triphosphate on electrical properties of subconfluent Madin-Darby canine kidney cells.

1. The present study has been performed to test for an influence of extracellular ATP on the potential differences across the cell membrane (PD) in subconfluent MDCK cells utilizing conventional microelectrodes. 2. In the absence of ATP, the mean measured PD was -47.5 +/- 0.3 mV (+/- S.E.M., n = 320). Application of 10 mumol/l ATP leads to rapid (less than 2 s) hyperpolarization of the cell membrane by -18.5 +/- 0.4 mV (n = 221), reduction of input resistance by 14 +/- 1 M omega (n = 106) and increase of the sensitivity of PD to alterations of extracellular potassium. 3. The concentration needed for half-maximal effect (K1/2) of ATP is approximately 0.5 mumol/l. ATP-gamma-S (K1/2 approximately 0.4 mumol/l) aand ADP (K1/2 approximately 0.9 similarly effective, whereas up to 1 mmol/l AMP or adenosine does not significantly alter PD. Application of 10 mumol/l theophylline, 1 mumol/l phentolamine and 10 mumol/l indomethacin does not blunt the hyperpolarizing effect of ATP. 4. The ATP-induced hyperpolarization is completely abolished in the presence of 1 mmol/l quinidine but only incompletely by 0.1 mmol/l quinidine or 1 mmol/l barium. In calcium-free extracellular fluid (1 mmol/l EDTA added) PD is 18.5 +/- 1.7 mV (n = 18). With reduced extracellular calcium, the hyperpolarizing effect of ATP is blunted (-12.3 +/- 1.6 mV, n = 18) and only transient. 5. In conclusion, ATP hyperpolarizes MDCK cells by increasing the potassium conductance. The activation of potassium channels requires calcium.