[Ca2+]i Oscillations and IL-6 Release Induced by α-Hemolysin from Escherichia coli Require P2 Receptor Activation in Renal Epithelia

Urinary tract infections are commonly caused by α-hemolysin (HlyA)-producing Escherichia coli. In erythrocytes, the cytotoxic effect of HlyA is strongly amplified by P2X receptors, which are activated by extracellular ATP released from the cytosol of the erythrocytes. In renal epithelia, HlyA causes reversible [Ca²⁺]_i oscillations, which trigger interleukin-6 (IL-6) and IL-8 release. We speculate that this effect is caused by HlyA-induced ATP release from the epithelial cells and successive P2 receptor activation. Here, we demonstrate that HlyA-induced [Ca²⁺]_i oscillations in renal epithelia were completely prevented by scavenging extracellular ATP. In accordance, HlyA was unable to inflict any [Ca²⁺]_i oscillations in 132-1N1 cells, which lack P2R completely. After transfecting these cells with the hP2Y₂ receptor, HlyA readily triggered [Ca²⁺]_i oscillations, which were abolished by P2 receptor antagonists. Moreover, HlyA-induced [Ca²⁺]_i oscillations were markedly reduced in medullary thick ascending limbs isolated from P2Y₂ receptor-deficient mice compared with wild type. Interestingly, the following HlyA-induced IL-6 release was absent in P2Y₂ receptor-deficient mice. This suggests that HlyA induces ATP release from renal epithelia, which via P2Y₂ receptors is the main mediator of HlyA-induced [Ca²⁺]_i oscillations and IL-6 release. This supports the notion that ATP signaling occurs early during bacterial infection and is a key player in the further inflammatory response.     

Ca influx and ATP release mediated by mechanical stretch in human lung fibroblasts

One cause of progressive pulmonary fibrosis is dysregulated wound healing after lung inflammation or damage in patients with idiopathic pulmonary fibrosis and severe acute respiratory distress syndrome. The mechanical forces are considered to regulate pulmonary fibrosis via activation of lung fibroblasts. In this study, the effects of mechanical stretch on the intracellular Ca²⁺ concentration ([Ca²⁺]_i) and ATP release were investigated in primary human lung fibroblasts. Uniaxial stretch (10–30% in strain) was applied to fibroblasts cultured in a silicone chamber coated with type I collagen using a stretching apparatus. Following stretching and subsequent unloading, [Ca²⁺]_i transiently increased in a strain-dependent manner. Hypotonic stress, which causes plasma membrane stretching, also transiently increased the [Ca²⁺]_i. The stretch-induced [Ca²⁺]_i elevation was attenuated in Ca²⁺-free solution. In contrast, the increase of [Ca²⁺]_i by a 20% stretch was not inhibited by the inhibitor of stretch-activated channels GsMTx-4, Gd³⁺, ruthenium red, or cytochalasin D. Cyclic stretching induced significant ATP releases from fibroblasts. However, the stretch-induced [Ca²⁺]_i elevation was not inhibited by ATP diphosphohydrolase apyrase or a purinergic receptor antagonist suramin. Taken together, mechanical stretch induces Ca²⁺ influx independently of conventional stretch-sensitive ion channels, the actin cytoskeleton, and released ATP.     

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.     

Increase of intracellular Ca2+ by adenine and uracil nucleotides in human midbrain-derived neuronal progenitor cells

Nucleotides play an important role in brain development and may exert their action via ligand-gated cationic channels or G protein-coupled receptors. Patch-clamp measurements indicated that in contrast to AMPA, ATP did not induce membrane currents in human midbrain derived neuronal progenitor cells (hmNPCs). Various nucleotide agonists concentration-dependently increased [Ca²⁺]_i as measured by the Fura-2 method, with the rank order of potency ATP > ADP > UTP > UDP. A Ca²⁺-free external medium moderately decreased, whereas a depletion of the intracellular Ca²⁺ storage sites by cyclopiazonic acid markedly depressed the [Ca²⁺]_i transients induced by either ATP or UTP. Further, the P2Y₁ receptor antagonistic PPADS and MRS 2179, as well as the nucleotide catalyzing enzyme apyrase, allmost abolished the effects of these two nucleotides. However, the P2Y_1,2,12 antagonistic suramin only slightly blocked the action of ATP, but strongly inhibited that of UTP. In agreement with this finding, UTP evoked the release of ATP from hmNPCs in a suramin-, but not PPADS-sensitive manner. Immunocytochemistry indicated the co-localization of P2Y_1,2,4-immunoreactivities (IR) with nestin-IR at these cells. In conclusion, UTP may induce the release of ATP from hmNPCs via P2Y₂ receptor-activation and thereby causes [Ca²⁺]_i transients by stimulating a P2Y₁-like receptor.     

Evidence for the possible involvement of the P2Y<Subscript>6</Subscript> receptor in Ca<Superscript>2+</Superscript> mobilization and insulin secretion in mouse pancreatic islets

Subtypes of purinergic receptors involved in modulation of cytoplasmic calcium ion concentration ([Ca²⁺]_i) and insulin release in mouse pancreatic β-cells were examined in two systems, pancreatic islets in primary culture and beta-TC6 insulinoma cells. Both systems exhibited some physiological responses such as acetylcholine-stimulated [Ca²⁺]_i rise via cytoplasmic Ca²⁺ mobilization. Addition of ATP, ADP, and 2-MeSADP (each 100 μM) transiently increased [Ca²⁺]_i in single islets cultured in the presence of 5.5 mM (normal) glucose. The potent P2Y₁ receptor agonist 2-MeSADP reduced insulin secretion significantly in islets cultured in the presence of high glucose (16.7 mM), whereas a slight stimulation occurred at 5.5 mM glucose. The selective P2Y₆ receptor agonist UDP (200 μM) transiently increased [Ca²⁺]_i and reduced insulin secretion at high glucose, whereas the P2Y_2/4 receptor agonist UTP and adenosine receptor agonist NECA were inactive. [Ca²⁺]_i transients induced by 2-MeSADP and UDP were antagonized by suramin (100 μM), U73122 (2 μM, PLC inhibitor), and 2-APB (10 or 30 μM, IP₃ receptor antagonist), but neither by staurosporine (1 μM, PKC inhibitor) nor depletion of extracellular Ca²⁺. The effect of 2-MeSADP on [Ca²⁺]_i was also significantly inhibited by MRS2500, a P2Y₁ receptor antagonist. These results suggested that P2Y₁ and P2Y₆ receptor subtypes are involved in Ca²⁺ mobilization from intracellular stores and insulin release in mouse islets. In beta-TC6 cells, ATP, ADP, 2-MeSADP, and UDP transiently elevated [Ca²⁺]_i and slightly decreased insulin secretion at normal glucose, while UTP and NECA were inactive. RT-PCR analysis detected mRNAs of P2Y₁ and P2Y₆, but not P2Y₂ and P2Y₄ receptors.     

The effects of extracellular nucleotides on [Ca2+]i signalling in a human‐derived renal proximal tubular cell line (HKC‐8)

HKC‐8 cells are a human‐derived renal proximal tubular cell line and provide a useful model system for the study of human renal cell function. In this study, we aimed to determine [Ca²⁺]_i signalling mediated by P2 receptor in HKC‐8. Fura‐2 and a ratio imaging method were employed to measure [Ca²⁺]_i in HKC‐8 cells. Our results showed that activation of P2Y receptors by ATP induced a rise in [Ca²⁺]_i that was dependent on an intracellular source of Ca²⁺, while prolonged activation of P2Y receptors induced a rise in [Ca²⁺]_i that was dependent on intra‐ and extracellular sources of Ca²⁺. Pharmacological and molecular data in this study suggests that TRPC4 channels mediate Ca²⁺ entry in coupling to activation of P2Y in HKC‐8 cells. U73221, an inhibitor of PI‐PLC, did not inhibit the initial ATP‐induced response; whereas D609, an inhibitor of PC‐PLC, caused a significant decrease in the initial ATP‐induced response, suggesting that P2Y receptors are coupled to PC‐PLC. Although P2X were present in HKC‐8, The P2X agonist, α,β me‐ATP, failed to cause a rise in [Ca²⁺]_i. However, PPADS at a concentration of 100 µM inhibits the ATP‐induced rise in [Ca²⁺]_i. Our results indicate the presence of functional P2Y receptors in HKC‐8 cells. ATP‐induced [Ca²⁺]_i elevation via P2Y is tightly associated with PC‐PLC and TRP channel. J. Cell. Biochem. 109: 132–139, 2010. © 2009 Wiley‐Liss, Inc.     

Lysosomal exocytosis of ATP is coupled to P2Y2 receptor in marginal cells in the stria vascular in neonatal rats

Adenosine triphosphate (ATP) is stored as lysosomal vesicles in marginal cells of the stria vascular in neonatal rats, but the mechanisms of ATP release are unclear. Primary cultures of marginal cells from 1-day-old Sprague–Dawley rats were established. P2Y₂ receptor and inositol 1,4,5-trisphosphate (IP3) receptor were immunolabelled in marginal cells of the stria vascular. We found that 30 μM ATP and 30 μM uridine triphosphate (UTP) evoked comparable significant increases in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in the absence of extracellular Ca²⁺, whereas the response was suppressed by 100 μM suramin, 10 μM 1-(6-(17β-3-methoxyester-1,3,5(10)-trien-17-yl)amino)-hexyl)-1H-pyrrole-2,5-dione(U-73122), 100 μM 2-aminoethoxydiphenyl borate (2-APB) and 5 μM thapsigargin (TG), thus indicating that ATP coupled with the P2Y₂R-PLC-IP3 pathway to evoke Ca²⁺ release from the endoplasmic reticulum (ER). Incubation with 200 μM Gly-Phe-β-naphthylamide (GPN) selectively disrupted lysosomes and caused significant increases in [Ca²⁺]_I; this effect was partly inhibited by P2Y₂R-PLC-IP3 pathway antagonists. After pre-treatment with 5 μM TG, [Ca²⁺]_i was significantly lower than that after treatment with P2Y₂R-PLC-IP3 pathway antagonists under the same conditions, thus indicating that lysosomal Ca²⁺ triggers Ca²⁺ release from ER Ca²⁺ stores. Baseline [Ca²⁺]_i declined after treatment with the Ca²⁺ chelator 50 μM bis-(aminophenolxy) ethane-N,N,Nʹ,Nʹ-tetra-acetic acid acetoxyme-thyl ester (BAPTA-AM) and 4 IU/ml apyrase. 30 μM ATP decrease of the number of quinacrine-positive vesicles via lysosome exocytosis, whereas the number of lysosomes did not change. However, lysosome exocytosis was significantly suppressed by pre-treatment with 5 μM vacuolin-1. Release of ATP and β-hexosaminidase both increased after treatment with 200 μM GPN and 5 μM TG, but decreased after incubation with 50 μM BAPTA-AM, 4 IU/ml apyrase and 5 μM vacuolin-1. We suggest that ATP triggers Ca²⁺ release from the ER, thereby contributing to secretion of lysosomal ATP via lysosomal exocytosis. Lysosomal stored Ca²⁺ triggers Ca²⁺ release from the ER directly though the IP3 receptors, and lysosomal ATP evokes Ca²⁺ signals indirectly via the P2Y₂R-PLC-IP3 pathway.     

ATP Released by Electrical Stimuli Elicits Calcium Transients and Gene Expression in Skeletal Muscle

ATP released from cells is known to activate plasma membrane P2X (ionotropic) or P2Y (metabotropic) receptors. In skeletal muscle cells, depolarizing stimuli induce both a fast calcium signal associated with contraction and a slow signal that regulates gene expression. Here we show that nucleotides released to the extracellular medium by electrical stimulation are partly involved in the fast component and are largely responsible for the slow signals. In rat skeletal myotubes, a tetanic stimulus (45 Hz, 400 1-ms pulses) rapidly increased extracellular levels of ATP, ADP, and AMP after 15 s to 3 min. Exogenous ATP induced an increase in intracellular free Ca²⁺ concentration, with an EC₅₀ value of 7.8 ± 3.1 μm. Exogenous ADP, UTP, and UDP also promoted calcium transients. Both fast and slow calcium signals evoked by tetanic stimulation were inhibited by either 100 μm suramin or 2 units/ml apyrase. Apyrase also reduced fast and slow calcium signals evoked by tetanus (45 Hz, 400 0.3-ms pulses) in isolated mouse adult skeletal fibers. A likely candidate for the ATP release pathway is the pannexin-1 hemichannel; its blockers inhibited both calcium transients and ATP release. The dihydropyridine receptor co-precipitated with both the P2Y₂ receptor and pannexin-1. As reported previously for electrical stimulation, 500 μm ATP significantly increased mRNA expression for both c-fos and interleukin 6. Our results suggest that nucleotides released during skeletal muscle activity through pannexin-1 hemichannels act through P2X and P2Y receptors to modulate both Ca²⁺ homeostasis and muscle physiology.     

Pleiotropic Effects of Bitter Taste Receptors on [Ca2+]i Mobilization,Hyperpolarization, and Relaxation of Human Airway Smooth Muscle Cells

Asthma is characterized by airway inflammation and airflow obstruction from human airway smooth muscle (HASM) constriction due to increased local bronchoconstrictive substances. We have recently found bitter taste receptors (TAS2Rs) on HASM, which increase [Ca²⁺]_i and relax the muscle. We report here that some, but not all, TAS2R agonists decrease [Ca²⁺]_i and relax HASM contracted by G-protein coupled receptors (GPCRs) that stimulate [Ca²⁺]_i. This suggests both a second pathway by which TAS2Rs relax, and, a heterogeneity of the response phenotype. We utilized eight TAS2R agonists and five procontractile GPCR agonists in cultured HASM cells. We find that heterogeneity in the inhibitory response hinges on which procontractile GPCR is activated. For example, chloroquine inhibits [Ca²⁺]_i increases from histamine, but failed to inhibit [Ca²⁺]_i increases from endothelin-1. Conversely, aristolochic acid inhibited [Ca²⁺]_i increases from endothelin-1 but not histamine. Other dichotomous responses were found when [Ca²⁺]_i was stimulated by bradykinin, angiotensin, and acetylcholine. There was no association between [Ca²⁺]_i inhibition and TAS2R subtype, nor whether [Ca²⁺]_i was increased by G_q- or G_i-coupled GPCRs. Selected studies revealed a correlation between [Ca²⁺]_i inhibition and HASM cell-membrane hyperpolarization. To demonstrate physiologic correlates, ferromagnetic beads were attached to HASM cells and cell stiffness measured by magnetic twisting cytometry. Consistent with the [Ca²⁺]_i inhibition results, chloroquine abolished the cell stiffening response (contraction) evoked by histamine but not by endothelin-1, while aristolochic acid inhibited cell stiffening from endothelin-1, but not from histamine. In studies using intact human bronchi, these same differential responses were found. Those TAS2R agonists that decreased [Ca²⁺]_i, promoted hyperpolarization, and decreased HASM stiffness, caused relaxation of human airways. Thus TAS2Rs relax HASM in two ways: a low-efficiency de novo [Ca²⁺]_i stimulation, and, a high-efficiency inhibition of GPCR-stimulated [Ca²⁺]_i. Furthermore, there is an interaction between TAS2Rs and some GPCRs that facilitates this [Ca²⁺]_i inhibition limb.     

Glycolytic ATP Fuels the Plasma Membrane Calcium Pump Critical for Pancreatic Cancer Cell Survival

Pancreatic cancer is an aggressive cancer with poor prognosis and limited treatment options. Cancer cells rapidly proliferate and are resistant to cell death due, in part, to a shift from mitochondrial metabolism to glycolysis. We hypothesized that this shift is important in regulating cytosolic Ca²⁺ ([Ca²⁺]_i), as the ATP-dependent plasma membrane Ca²⁺ ATPase (PMCA) is critical for maintaining low [Ca²⁺]_i and thus cell survival. The present study aimed to determine the relative contribution of mitochondrial versus glycolytic ATP in fuelling the PMCA in human pancreatic cancer cells. We report that glycolytic inhibition induced profound ATP depletion, PMCA inhibition, [Ca²⁺]_i overload, and cell death in PANC1 and MIA PaCa-2 cells. Conversely, inhibition of mitochondrial metabolism had no effect, suggesting that glycolytic ATP is critical for [Ca²⁺]_i homeostasis and thus survival. Targeting the glycolytic regulation of the PMCA may, therefore, be an effective strategy for selectively killing pancreatic cancer while sparing healthy cells.     

Extracellular NAD induces a rise in [Ca]i in activated human monocytes via engagement of P2Y1 and P2Y11 receptors

Extracellular nicotinamide adenine dinucleotide (NAD⁺) is known to increase the intracellular calcium concentration [Ca²⁺]_i in different cell types and by various mechanisms. Here we show that NAD⁺ triggers a transient rise in [Ca²⁺]_i in human monocytes activated with lipopolysaccharide (LPS), which is caused by a release of Ca²⁺ from IP₃-responsive intracellular stores and an influx of extracellular Ca²⁺. By the use of P2 receptor-selective agonists and antagonists we demonstrate that P2 receptors play a role in the NAD⁺-induced calcium response in activated monocytes. Of the two subclasses of P2 receptors (P2X and P2Y) the P2Y receptors were considered the most likely candidates, since they share calcium signaling properties with NAD⁺. The identification of P2Y₁ and P2Y₁₁ as receptor subtypes responsible for the NAD⁺-triggered increase in [Ca²⁺]_i was supported by several lines of evidence. First, specific P2Y₁ and P2Y₁₁ receptor antagonists inhibited the NAD⁺-induced increase in [Ca²⁺]_i. Second, NAD⁺ was shown to potently induce calcium signals in cells transfected with either subtype, whereas untransfected cells were unresponsive. Third, NAD⁺ caused an increase in [cAMP]_i, prevented by the P2Y₁₁ receptor-specific antagonist NF157.     

Two Distinct P2 Purinergic Receptors, P2Y and P2U, Are Coupled to Phospholipase C in Mouse Pineal Gland Tumor Cells

Abstract: We found that extracellular ATP can increase the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in mouse pineal gland tumor (PGT-β) cells. Studies of the [Ca²⁺]_i rise using nucleotides and ATP analogues established the following potency order: ATP, adenosine 5′-O-(3-thiotriphosphate) ≥ UTP > 2-chloro-ATP > 3′-O-(4-benzoyl)benzoyl ATP, GTP ≥ 2-methylthio ATP, adenosine 5′-O-(2-thiodiphosphate) (ADPβS) > CTP. AMP, adenosine, α,β-methyleneadenosine 5′-triphosphate, β,γ-methyleneadenosine 5′-triphosphate, and UMP had little or no effect on the [Ca²⁺]_i rise. Raising the extracellular Mg²⁺ concentration to 10 mM decreases the ATP-and UTP-induced [Ca²⁺]_i rise, because the responses depend on the ATP^4? and UTP^4? concentrations, respectively. The P_2U purinoceptor-selective agonist UTP and the P_2Y purinoceptor-selective agonist ADPβS induce inositol 1,4,5-trisphosphate generation in a concentration-dependent manner with maximal effective concentrations of ～100 µM. In sequential stimulation, UTP and ADPβS do not interfere with each other in raising the [Ca²⁺]_i. Costimulation with UTP and ADPβS results in additive inositol 1,4,5-trisphosphate generation to a similar extent as is achieved with ATP alone. Pretreatment with pertussis toxin inhibits the action of UTP and ATP by maximally 45–55%, whereas it has no effect on the ADPβS response. Treatment with 1 µM phorbol 12-myristate 13-acetate inhibits the ADPβS-induced [Ca²⁺]_i rise more effectively than the ATP- and UTP-induced responses. These results suggest that P_2U and P_2Y purinoceptors coexist on PGT-β cells and that both receptors are linked to phospholipase C.     

Ca regulation of connexin 43 hemichannels in C6 glioma and glial cells

Connexin hemichannels have a low open probability under normal conditions but open in response to various stimuli, forming a release pathway for small paracrine messengers. We investigated hemichannel-mediated ATP responses triggered by changes of intracellular Ca²⁺ ([Ca²⁺]_i) in Cx43 expressing glioma cells and primary glial cells. The involvement of hemichannels was confirmed with gja1 gene-silencing and exclusion of other release mechanisms. Hemichannel responses were triggered when [Ca²⁺]_i was in the 500 nM range but the responses disappeared with larger [Ca²⁺]_i transients. Ca²⁺-triggered responses induced by A23187 and glutamate activated a signaling cascade that involved calmodulin (CaM), CaM-dependent kinase II, p38 mitogen activated kinase, phospholipase A2, arachidonic acid (AA), lipoxygenases, cyclo-oxygenases, reactive oxygen species, nitric oxide and depolarization. Hemichannel responses were also triggered by activation of CaM with a Ca²⁺-like peptide or exogenous application of AA, and the cascade was furthermore operational in primary glial cells isolated from rat cortex. In addition, several positive feed-back loops contributed to amplify the responses. We conclude that an elevation of [Ca²⁺]_i triggers hemichannel opening, not by a direct action of Ca²⁺ on hemichannels but via multiple intermediate signaling steps that are adjoined by distinct signaling mechanisms activated by high [Ca²⁺]_i and acting to restrain cellular ATP loss.     

Cell density‐dependent changes in intracellular Ca2+ mobilization via the P2Y2 receptor in rat bone marrow stromal cells

Bone marrow stromal cells (BMSCs) are an interesting subject of research because they have characteristics of mesenchymal stem cells. We investigated intracellular Ca²⁺ signaling in rat BMSCs. Agonists for purinergic receptors increased intracellular Ca²⁺ levels ([Ca²⁺]_i). The order of potency followed ATP = UTP > ADP = UDP. ATP‐induced rise in [Ca²⁺]_i was suppressed by U73122 and suramin, but not by pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulfonic acid (PPADS), suggesting the functional expression of G protein‐coupled P2Y₂ receptors. RT‐PCR and immunohistochemical studies also showed the expression of P2Y₂ receptors. [Ca²⁺]_i response to UTP changed with cell density. The UTP‐induced rise in [Ca²⁺]_i was greatest at high density. V_max (maximum Ca²⁺ response) and EC₅₀ (agonist concentration that evokes 50% of V_max) suggest that the amount and property of P2Y₂ receptors were changed by cell density. Note that UTP induced Ca²⁺ oscillation at only medium cell density. Pharmacological studies indicated that UTP‐induced Ca²⁺ oscillation required Ca²⁺ influx by store‐operated Ca²⁺ entry. Carbenoxolone, a gap junction blocker, enhanced Ca²⁺ oscillation. Immunohistochemical and quantitative real‐time PCR studies revealed that proliferating cell nuclear antigen (PCNA)‐positive cells declined but the mRNA expression level of the P2Y₂ receptor increased as cell density increased. Co‐application of fetal calf serum with UTP induced Ca²⁺ oscillation at high cell density. These results suggest that the different patterns observed for [Ca²⁺]_i mobilization with respect to cell density may be associated with cell cycle progression. J. Cell. Physiol. 219: 372–381, 2009. © 2009 Wiley‐Liss, Inc.     

Changes in Intracellular Ca2+ and Energy Levels During In Vitro Ischemia in the Gerbil Hippocampal Slice

Abstract: The time course of the decline in energy levels during an in vitro ischemia-like condition was compared with changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in subregions of the gerbil hippocampal slice [CA1, CA3, and the inner and outer portions of the dentate gyrus (DG)]. Hippocampal transverse slices were loaded with a fluorescent indicator, rhod-2. During the on-line monitoring of [Ca²⁺]_i, the slices were perfused with an in vitro ischemia-like medium (33°C). The slices were collected at several experimental time points, frozen, dried, and dissected into subregions. The contents of adenine nucleotides (ATP, ADP, and AMP) and phosphocreatine (PCr) were measured by HPLC methods. Region-specific and acute [Ca²⁺]_i elevations were observed in CA1 ～4 min after onset of the in vitro ischemia-like condition and also in the inner portion of the DG with a delay of 10–40 s. The change in ATP levels was related to the increase in [Ca²⁺]_i. ATP levels in all subregions gradually decreased before the acute [Ca²⁺]_i elevation. Concomitant with the acute [Ca²⁺]_i elevation in CA1 and the inner portion of the DG, ATP levels in the subregions rapidly decreased, whereas declines in levels of high-energy-charge phosphates were gradual in CA3 and the outer portion of the DG, in which the remarkable [Ca²⁺]_i elevation was not observed. These results suggest that ATP depletion observed in CA1 and the inner portion of the DG is due to the region-specific increase in [Ca²⁺]_i, which activates a Ca²⁺-ATP-driven pump and produces a subsequent fall in neuronal ATP content.     

Permeation of Calcium through Purified Connexin 26 Hemichannels

Gap junction channels communicate the cytoplasms of two cells and are formed by head to head association of two hemichannels, one from each of the cells. Gap junction channels and hemichannels are permeable to ions and hydrophilic molecules of up to M_r 1,000, including second messengers and metabolites. Intercellular Ca²⁺ signaling can occur by movement of a number of second messengers, including Ca²⁺, through gap junction channels, or by a paracrine pathway that involves activation of purinergic receptors in neighboring cells following ATP release through hemichannels. Understanding Ca²⁺ permeation through Cx26 hemichannels is important to assess the role of gap junction channels and hemichannels in health and disease. In this context, it is possible that increased Ca²⁺ influx through hemichannels under ischemic conditions contributes to cell damage. Previous studies suggest Ca²⁺ permeation through hemichannels, based on indirect arguments. Here, we demonstrate for the first time hemichannel permeability to Ca²⁺ by measuring Ca²⁺ transport through purified Cx26 hemichannels reconstituted in liposomes. We trapped the low affinity Ca²⁺-sensitive fluorescent probe Fluo-5N into the liposomes and followed the increases in intraliposomal [Ca²⁺] in response to an imposed [Ca²⁺] gradient. We show that Ca²⁺ does move through Cx26 hemichannels and that the permeability of the hemichannels to Ca²⁺ is high, similar to that for Na⁺. We suggest that hemichannels can be a significant pathway for Ca²⁺ influx into cells under conditions such as ischemia.     

The Plasma Membrane Calcium Pump in Pancreatic Cancer Cells Exhibiting the Warburg Effect Relies on Glycolytic ATP

Evidence suggests that the plasma membrane Ca²⁺-ATPase (PMCA), which is critical for maintaining a low intracellular Ca²⁺ concentration ([Ca²⁺]_i), utilizes glycolytically derived ATP in pancreatic ductal adenocarcinoma (PDAC) and that inhibition of glycolysis in PDAC cell lines results in ATP depletion, PMCA inhibition, and an irreversible [Ca²⁺]_i overload. We explored whether this is a specific weakness of highly glycolytic PDAC by shifting PDAC cell (MIA PaCa-2 and PANC-1) metabolism from a highly glycolytic phenotype toward mitochondrial metabolism and assessing the effects of mitochondrial versus glycolytic inhibitors on ATP depletion, PMCA inhibition, and [Ca²⁺]_i overload. The highly glycolytic phenotype of these cells was first reversed by depriving MIA PaCa-2 and PANC-1 cells of glucose and supplementing with α-ketoisocaproate or galactose. These culture conditions resulted in a significant decrease in both glycolytic flux and proliferation rate, and conferred resistance to ATP depletion by glycolytic inhibition while sensitizing cells to mitochondrial inhibition. Moreover, in direct contrast to cells exhibiting a high glycolytic rate, glycolytic inhibition had no effect on PMCA activity and resting [Ca²⁺]_i in α-ketoisocaproate- and galactose-cultured cells, suggesting that the glycolytic dependence of the PMCA is a specific vulnerability of PDAC cells exhibiting the Warburg phenotype.     

Purinergic P2X7 Receptors Mediate ATP-induced Saliva Secretion by the Mouse Submandibular Gland

Salivary glands express multiple isoforms of P2X and P2Y nucleotide receptors, but their in vivo physiological roles are unclear. P2 receptor agonists induced salivation in an ex vivo submandibular gland preparation. The nucleotide selectivity sequence of the secretion response was BzATP ≫ ATP > ADP ≫ UTP, and removal of external Ca²⁺ dramatically suppressed the initial ATP-induced fluid secretion (∼85%). Together, these results suggested that P2X receptors are the major purinergic receptor subfamily involved in the fluid secretion process. Mice with targeted disruption of the P2X₇ gene were used to evaluate the role of the P2X₇ receptor in nucleotide-evoked fluid secretion. P2X₇ receptor protein and BzATP-activated inward cation currents were absent, and importantly, purinergic receptor agonist-stimulated salivation was suppressed by more than 70% in submandibular glands from P2X₇-null mice. Consistent with these observations, the ATP-induced increases in [Ca²⁺]_i were nearly abolished in P2X₇^–/– submandibular acinar and duct cells. ATP appeared to also act through the P2X₇ receptor to inhibit muscarinic-induced fluid secretion. These results demonstrate that the ATP-sensitive P2X₇ receptor regulates fluid secretion in the mouse submandibular gland.Salivation is a Ca²⁺-dependent process (1, 2) primarily associated with the neurotransmitters norepinephrine and acetylcholine, release of which stimulates α-adrenergic and muscarinic receptors, respectively. Both types of receptors are coupled to G proteins that activate phospholipase Cβ (PLCβ) during salivary gland stimulation. PLCβ activation cleaves phosphatidylinositol 1,4-bisphosphate resulting in diacylglycerol and inositol 1,4,5-trisphosphate (InsP₃) production. Activation of Ca²⁺-selective InsP₃ receptor channels localized to the endoplasmic reticulum of salivary acinar cells increases the intracellular free calcium concentration ([Ca²⁺]_i).⁴ Depletion of the endoplasmic reticulum Ca²⁺ pool triggers extracellular Ca²⁺ influx and a sustained elevation in [Ca²⁺]_i. This increase in [Ca²⁺]_i activates Ca²⁺-dependent K⁺ and Cl^– channels promoting Cl^– secretion across the apical membrane and a lumen negative, electrochemical gradient that supports Na⁺ efflux into the lumen. The accumulation of NaCl creates an osmotic gradient which drives water movement into the lumen, thus generating isotonic primary saliva. This primary fluid is then modified by the ductal system, which reabsorbs NaCl and secretes KHCO₃ producing a final saliva that is hypotonic (1, 2).Salivation also has a non-cholinergic, non-adrenergic component, the origin of which is unclear (3). In addition to muscarinic and α-adrenergic receptors, salivary acinar cells express other receptors that are coupled to an increase in [Ca²⁺]_i such as purinergic P2 and substance P receptors. Like muscarinic and α-adrenergic receptors, P2 receptor activation leads to a sustained increase in [Ca²⁺]_i in salivary acinar cells (4). In contrast, substance P receptor activation rapidly desensitizes and therefore generates only a relatively transient increase in [Ca²⁺]_i (5) that is unlikely to appreciably contribute to fluid secretion. The purinergic P2 receptor family is comprised of G protein-coupled P2Y and ionotropic P2X receptors activated by extracellular di- and triphosphate nucleotides. Activation of both subfamilies of P2 receptors causes an increase in [Ca²⁺]_i. P2Y receptors increase [Ca²⁺]_i via InsP₃-induced Ca²⁺ mobilization from intracellular stores (similar to α-adrenergic and muscarinic receptors) while P2X receptors act as ligand-gated, non-selective cation channels that mediate extracellular Ca²⁺ influx (6). Salivary gland tissues express at least four isoforms of P2X (P2X₄ and P2X₇) and P2Y (P2Y₁ and P2Y₂) subtypes; however, their in vivo physiological significance has yet to be characterized (7–11).Our results revealed that ATP acts in isolation to stimulate fluid secretion from the mouse submandibular gland, but secretion is inhibited when ATP is simultaneously presented with a muscarinic receptor agonist. Ablation of the P2X₇ gene had no effect on the salivary flow rate evoked by muscarinic receptor activation, but markedly reduced ATP-mediated fluid secretion and rescued the inhibitory effects of ATP on muscarinic receptor activation. Submandibular gland acinar cells from P2X₇^–/– animals had dramatically impaired ATP-activated Ca²⁺ signaling, consistent with this being the mechanism responsible for the reduction in ATP-mediated fluid secretion in these mice. Together, these results demonstrated that ATP regulates salivation, acting mainly through the P2X₇ receptor. Activation of the P2X₇ receptor may play a major role in non-adrenergic, non-cholinergic stimulated fluid secretion.     

Temperature dependency of calcium responses in mammary tumour cells

Changes of intracellular calcium concentration ([Ca²⁺]_i) induced by the extracellular application of ATP and bradykinin in mouse mammary tumour cells (MMT060562) were investigated by image analysis of fluo-3 fluorescence at 24°C and 35°C. ATP (0·1–100 μM ) and bradykinin (0·1 nM –1 μM ) induced the increase of [Ca²⁺]_i at both temperatures and Ca²⁺-depletion did not affect these [Ca²⁺]_i responses. Both [Ca²⁺]_i responses became more sensitive at 35°C than at 24°C. A clear latency of [Ca²⁺]_i increased after the application of the agonists was observed, and it changed with the concentration of the agonist. As concentrations of ATP or bradykinin became lower, the latency and rise time became longer. At higher concentrations, the latency and rise time approached a constant value. The latency shortened remarkably at 35°C. These results suggested the involvement of a regenerative or threshold process in the [Ca²⁺]_i responses in mammary tumour cells. © 1997 John Wiley & Sons, Ltd.     

Signal Transduction Pathways Coupled to a P2U Receptor in Neuroblastoma × Glioma (NG108-15) Cells

Abstract: Extracellular ATP has neurotransmitter-like properties in the CNS and PNS that are mediated by a cell-surface P₂ purinergic receptor. In the present study, we have extensively characterized the signal transduction pathways that are associated with activation of a P₂U receptor in a cultured neuroblastoma × glioma hybrid cell line (NG108-15 cells). The addition of ≥1 μM ATP to NG108-15 cells caused a transient increase in [Ca²⁺]_i that was inhibited by 40% when extracellular calcium was chelated by EGTA. ATP concentrations ≥500 μM also elicited a sustained increase in [Ca²⁺]_i that was inhibited when extracellular calcium was chelated by EGTA. The increase in [Ca²⁺]_i elicited by ATP occurred concomitantly with the hydrolysis off [³²P]-phosphatidylinositol 4,5-bisphosphates and an increase in the level of inositol 1,4,5-trisphosphate. ATP also caused a time- and dose-dependent increase in levels of [3H]inositol monophosphates in lithium-treated cells. Separation of the inositol monophosphate isomers by ion chromatography revealed a specific increase in the level of inositol 4-monophosphate. The magnitude of the increase in [Ca²⁺]_i elicited by ATP correlated with the concentration of the fully ionized form of ATP (ATP^4-) in the medium and not with the concentration of magnesium-ATP (MgATP^2-). Similar to ATP, UTP also induced polyphosphoinositide breakdown, inositol phosphate formation, and an increase in [Ca²⁺]_i. ADP, ITP, TTP, GTP, ATP-γS, 2-methylthio ATP, β,γ-imidoATP or 3′-O-(4-benzoyl)benzoylATP, but not CTP, AMP, β,γ-methylene ATP, or adenosine, also caused an increase in [Ca²⁺]_i. In cells labeled with [³²P]P_i or [¹⁴C]-arachidonic acid, ATP caused a transient increase in levels of labeled phosphatidic acids, but had no effect on levels of arachidonic acid. The increase in phosphatidic acid levels elicited by ATP apparently was not due to activation of a phospholipase D because ATP did not induce the formation of phosphatidylethanol in [¹⁴C]myristic acid-labeled cells incubated in the presence of ethanol. These findings support the hypothesis that a P₂ nucleotide receptor in NG108-15 cells is coupled to a signal transduction pathway involving the activation of a phospholipase C and a plasma membrane calcium channel, but not the activation of phospholipases A₂ and D.