Na/K-ATPase tethers phospholipase C and IP3 receptor into a calcium-regulatory complex

We have shown that the caveolar Na/K-ATPase transmits ouabain signals via multiple signalplexes. To obtain the information on the composition of such complexes, we separated the Na/K-ATPase from the outer medulla of rat kidney into two different fractions by detergent treatment and density gradient centrifugation. Analysis of the light fraction indicated that both PLC-gamma1 and IP3 receptors (isoforms 2 and 3, IP3R2 and IP3R3) were coenriched with the Na/K-ATPase, caveolin-1 and Src. GST pulldown assays revealed that the central loop of the Na/K-ATPase alpha1 subunit interacts with PLC-gamma1, whereas the N-terminus binds IP3R2 and IP3R3, suggesting that the signaling Na/K-ATPase may tether PLC-gamma1 and IP3 receptors together to form a Ca(2+)-regulatory complex. This notion is supported by the following findings. First, both PLC-gamma1 and IP3R2 coimmunoprecipitated with the Na/K-ATPase and ouabain increased this interaction in a dose- and time-dependent manner in LLC-PK1 cells. Depletion of cholesterol abolished the effects of ouabain on this interaction. Second, ouabain induced phosphorylation of PLC-gamma1 at Tyr(783) and activated PLC-gamma1 in a Src-dependent manner, resulting in increased hydrolysis of PIP2. It also stimulated Src-dependent tyrosine phosphorylation of the IP3R2. Finally, ouabain induced Ca(2+) release from the intracellular stores via the activation of IP3 receptors in LLC-PK1 cells. This effect required the ouabain-induced activation of PLC-gamma1. Inhibition of Src or depletion of cholesterol also abolished the effect of ouabain on intracellular Ca(2+).     

Endoplasmic reticulum Ca2+ dysregulation and endoplasmic reticulum stress following in vitro neuronal ischemia: role of Na+-K+-Cl- cotransporter

We investigated the role of Na(+)-K(+)-Cl(-) cotransporter (NKCC1) in conjunction with Na(+)/Ca(2+) exchanger (NCX) in disruption of endoplasmic reticulum (ER) Ca(2+) homeostasis and ER stress development in primary cortical neurons following in vitro ischemia. Oxygen-glucose deprivation (OGD) and reoxygenation (REOX) caused a rise in [Na(+)](cyt) which was accompanied by an elevation in [Ca(2+)](cyt). Inhibition of NKCC1 with its potent inhibitor bumetanide abolished the OGD/REOX-induced rise in [Na(+)](cyt) and [Ca(2+)](cyt). Moreover, OGD significantly increased Ca(2+)(ER) accumulation. Following REOX, a biphasic change in Ca(2+)(ER) occurred with an initial release of Ca(2+)(ER) which was sensitive to inositol 1,4,5-trisphosphate receptor (IP(3)R) inhibition and a subsequent refilling of Ca(2+)(ER) stores. Inhibition of NKCC1 activity with its inhibitor or genetic ablation prevented the release of Ca(2+)(ER). A similar result was obtained with inhibition of reversed mode operation of NCX (NCX(rev)). OGD/REOX also triggered a transient increase of glucose regulated protein 78 (GRP78), phospho-form of the alpha subunit of eukaryotic initiation factor 2 (p-eIF2alpha), and cleaved caspase 12 proteins. Pre-treatment of neurons with NKCC1 inhibitor bumetanide inhibited upregulation of GRP78 and attenuated the level of cleaved caspase 12 and p-eIF2alpha. Inhibition of NKCC1 reduced cytochrome C release and neuronal death. Taken together, these results suggest that NKCC1 and NCX(rev) may be involved in ischemic cell damage in part via disrupting ER Ca(2+) homeostasis and ER function.     

Evidence on the operation of ATP-induced capacitative calcium entry in breast cancer cells and its blockade by 17beta-estradiol

Little is known about the regulation of cytosolic calcium Ca(2+) levels ([Ca(2+)](i)) in breast cancer cells. We investigated the existence of capacitative calcium entry (CCE) in the tumorigenic cell line MCF-7 and its responsiveness to ATP. MCF-7 cells express purinergic receptors as well as estrogen receptors (ER). Depletion of calcium stores with thapsigargin (TG, 500 nM) or ATP (10 microM) in the absence of extracellular Ca(2+), resulted in a rapid and transient elevation in [Ca(2+)](i). After recovery of basal levels, Ca(2+) readmission (1.5 mM) to the medium increased Ca(2+) influx (twofold over basal), reflecting pre-activation of a CCE pathway. Cells pretreated with TG were unable to respond to ATP, thus indicating that the same Ca(2+) store is involved in their response. Moreover, IP(3)-dependent ATP-induced calcium mobilization and CCE were completely blocked using compound U-73122, an inhibitor of phospholipase C. Compound 2-APB (75 microM) and Gd(3+) (10 microM), antagonists of the CCE pathway, completely prevented ATP-stimulated capacitative Ca(2+) entry. CCE in MCF-7 cells was highly permeable to Mn(2+) and to the Ca(2+) surrogate Sr(2+). Mn(2+) entry sensitivity to Gd(3+) matched that of the Ca(2+) entry pathway. 17Beta-estradiol blocked ATP-induced CCE, but was without effect on TG-induced CCE. Besides, the estrogen blockade of the ATP-induced CCE was completely abolished by preincubation of the cells with an ER monoclonal antibody. ER alpha immunoreactivity could also be detected in a purified plasma membrane fraction of MCF-7 cells. These results represent the first evidence on the operation of a ATP-responsive CCE pathway in MCF-7 cells and also indicate that 17beta-estradiol interferes with this mechanism by acting at the cell surface level.     

Co-activation of P2Y2 Receptor and TRPV Channel by ATP: Implications for ATP Induced Pain

1. Extracellular ATP is recognized as a peripheral modulator of pain. Activation of ionotropic P2X receptors in sensory neurons has been implicated in induction of pain, whereas metabotropic P2Y receptors in potentiation of pain induced by chemical or physical stimuli via capsaicin sensitive TRPV1 channel. Here we report that P2Y2 receptor activation by ATP can activate the TRPV1 channel in absence of any other stimuli. 2. ATP-induced Ca2+ signaling was studied in Neuro2a cells. ATP evoked release of intracellular Ca2+ from ER and Ca2+ influx through a fast inactivating channel. The Ca2+ response was induced by P2Y receptor agonists in the order of potency ATP>or=UTP>or=ATPgammaS>ADP and was inhibited by suramin and PPADS. The P2X receptor agonist alpha beta methyl ATP was ineffective. 3. The Ca2+ influx was blocked by ruthenium red, an inhibitor of TRPV1 channel. Capsaicin, the most potent activator of the TRPV1 channel, evoked a fast inactivating Ca2+ transient suggesting the presence of endogenous TRPV1 channels in Neuro2a cells. NMS and PDBu, repressors of IP3 formation, drastically inhibited both the components of Ca2+ response. 4. Our data show co-activation of the P2Y2 receptor and capsaicin sensitive TRPV1 channel by ATP. Such functional interaction between endogenous P2Y2 receptor and TRPV1 channels could explain the ATP-induced pain.     

Plasma membrane-cytoskeleton-endoplasmic reticulum complexes in neurons and astrocytes

The possibility that certain integral plasma membrane (PM) proteins involved in Ca(2+) homeostasis form junctional units with adjacent endoplasmic reticulum (ER) in neurons and glia was explored using immunoprecipitation and immunocytochemistry. Rat brain membranes were solubilized with the mild, non-ionic detergent, IGEPAL CA-630. Na(+)/Ca(2+) exchanger type 1 (NCX1), a key PM Ca(2+) transporter, was immunoprecipitated from the detergent-soluble fraction. Several abundant PM proteins co-immunoprecipitated with NCX1, including the alpha2 and alpha3 isoforms of the Na(+) pump catalytic (alpha) subunit, and the alpha2 subunit of the dihydropyridine receptor. The adaptor protein, ankyrin 2 (Ank 2), and the cytoskeletal proteins, alpha-fodrin and beta-spectrin, also selectively co-immunoprecipitated with NCX1, as did the ER proteins, Ca(2+) pump type 2 (SERCA 2), and inositol-trisphosphate receptor type 1 (IP(3)R-1). In contrast, a number of other abundant PMs, adaptors, and cytoskeletal proteins did not co-immunoprecipitate with NCX1, including the Na(+) pump alpha1 isoform, PM Ca(2+) pump type 1 (PMCA1), beta-fodrin, and Ank 3. In reciprocal experiments, immunoprecipitation with antibodies to the Na(+) pump alpha2 and alpha3 isoforms, but not alpha1, co-immunoprecipitated NCX1; the antibodies to alpha1 did, however, co-immunoprecipitate PMCA1. Antibodies to Ank 2, alpha-fodrin, beta-spectrin and IP(3)R-1 all co-immunoprecipitated NCX1. Immunocytochemistry revealed partial co-localization of beta-spectrin with NCX1, Na(+) pump alpha3, and IP(3)R-1 in neurons and of alpha-fodrin with NCX1 and SERCA2 in astrocytes. The data support the idea that in neurons and glia PM microdomains containing NCX1 and Na(+) pumps with alpha2 or alpha3 subunits form Ca(2+) signaling complexes with underlying ER containing SERCA2 and IP(3)R-1. These PM and ER components appear to be linked through the cytoskeletal spectrin network, to which they are probably tethered by Ank 2.     

The effect of dicyclohexylcarbodiimide and cyclopiazonic acid on the difference FTIR spectra of sarcoplasmic reticulum induced by photolysis of caged-ATP and caged-Ca2+.

The photochemical release of Ca2+ from caged-Ca2+ in the absence of ATP, and the release of ATP from caged-ATP in the presence of Ca2+ induce characteristic difference FTIR spectra on rabbit sarcoplasmic reticulum that are related to the formation of Ca2-E1 and E approximately P intermediates of the Ca(2+)-ATPase, respectively. Dicyclohexylcarbodiimide (10 nmol/mg protein) abolished both the Ca(2+)-and ATP-induced difference FTIR spectra parallel with inhibition of ATPase activity. Cyclopiazonic acid (50 nmol/mg protein) inhibited the Ca(2+)-induced difference spectrum measured in the absence of ATP, but had no significant effect on the ATP-induced difference spectrum measured in the presence of 1 mM Ca2+. The dog kidney Na+,K(+)-ATPase did not give significant difference spectrum after photolysis of caged-ATP in Ca(2+)-free media containing 90 mM Na+ and 10 mM K+, with or without ouabain. We propose that both the Ca2+ and the ATP-induced difference FTIR spectra of the Ca(2+)-ATPase reflect the occupancy of the high-affinity Ca2+ transport site of the enzyme.     

Bile acids induce Ca2+ release from both the endoplasmic reticulum and acidic intracellular calcium stores through activation of inositol trisphosphate receptors and ryanodine receptors

Gallstones can cause acute pancreatitis, an often fatal disease in which the pancreas digests itself. This is probably because of biliary reflux into the pancreatic duct and subsequent bile acid action on the acinar cells. Because Ca(2+) toxicity is important for the cellular damage in pancreatitis, we have studied the mechanisms by which the bile acid taurolithocholic acid 3-sulfate (TLC-S) liberates Ca(2+). Using two-photon plasma membrane permeabilization and measurement of [Ca(2+)] inside intracellular stores at the cell base (dominated by ER) and near the apex (dominated by secretory granules), we have characterized the Ca(2+) release pathways. Inhibition of inositol trisphosphate receptors (IP(3)Rs), by caffeine and 2-APB, reduced Ca(2+) release from both the ER and an acidic pool in the granular area. Inhibition of ryanodine receptors (RyRs) by ruthenium red (RR) also reduced TLC-S induced liberation from both stores. Combined inhibition of IP(3)Rs and RyRs abolished Ca(2+) release. RyR activation depends on receptors for nicotinic acid adenine dinucleotide phosphate (NAADP), because inactivation by a high NAADP concentration inhibited release from both stores, whereas a cyclic ADPR-ribose antagonist had no effect. Bile acid-elicited intracellular Ca(2+) liberation from both the ER and the apical acidic stores depends on both RyRs and IP(3)Rs.     

Hypoxia leads to Na,K-ATPase downregulation via Ca(2+) release-activated Ca(2+) channels and AMPK activation

To maintain cellular ATP levels, hypoxia leads to Na,K-ATPase inhibition in a process dependent on reactive oxygen species (ROS) and the activation of AMP-activated kinase α1 (AMPK-α1). We report here that during hypoxia AMPK activation does not require the liver kinase B1 (LKB1) but requires the release of Ca(2+) from the endoplasmic reticulum (ER) and redistribution of STIM1 to ER-plasma membrane junctions, leading to calcium entry via Ca(2+) release-activated Ca(2+) (CRAC) channels. This increase in intracellular Ca(2+) induces Ca(2+)/calmodulin-dependent kinase kinase β (CaMKKβ)-mediated AMPK activation and Na,K-ATPase downregulation. Also, in cells unable to generate mitochondrial ROS, hypoxia failed to increase intracellular Ca(2+) concentration while a STIM1 mutant rescued the AMPK activation, suggesting that ROS act upstream of Ca(2+) signaling. Furthermore, inhibition of CRAC channel function in rat lungs prevented the impairment of alveolar fluid reabsorption caused by hypoxia. These data suggest that during hypoxia, calcium entry via CRAC channels leads to AMPK activation, Na,K-ATPase downregulation, and alveolar epithelial dysfunction.     

Increase of [Ca(2+)](i) via activation of ATP receptors in PC-Cl3 rat thyroid cell line.

In PC-Cl3 rat thyroid cell line, ATP and UTP provoked a transient increase in [Ca(2+)](i), followed by a lower sustained phase. Removal of extracellular Ca(2+) reduced the initial transient response and completely abolished the plateau phase. Thapsigargin (TG) caused a rapid rise in [Ca(2+)](i) and subsequent addition of ATP was without effect. The transitory activation of [Ca(2+)](i) was dose-dependently attenuated in cells pretreated with the specific inhibitor of phospholipase C (PLC), U73122. These data suggest that the ATP-stimulated increment of [Ca(2+)](i) required InsP(3) formation and binding to its specific receptors in Ca(2+) stores. Desensitisation was demonstrated with respect to the calcium response to ATP and UTP in Fura 2-loaded cells. Further studies were performed to investigate whether the effect of ATP on Ca(2+) entry into PC-Cl3 cells was via L-type voltage-dependent Ca(2+) channels (L-VDCC) and/or by the capacitative pathway. Nifedipine decreased ATP-induced increase on [Ca(2+)](i). Addition of 2 mM Ca(2+) induced a [Ca(2+)](i) rise after pretreatment of the cells with TG or with 100 microM ATP in Ca(2+)-free medium. These data indicate that Ca(2+) entry into PC-Cl3 stimulated with ATP occurs through both an L-VDCC and through a capacitative pathway. Using buffers with differing Na(+) concentrations, we found that the effects of ATP were dependent of extracellular Na(+), suggesting that a Na(+)/Ca(2+) exchange mechanism is also operative. These data suggest the existence, in PC-Cl3 cell line, of a P2Y purinergic receptor able to increase the [Ca(2+)](i) via PLC activation, Ca(2+) store depletion, capacitative Ca(2+) entry and L-VDCC activation.     

Interplay between ryanodine and IP3 receptors in ATP-stimulated mouse luteinized-granulosa cells

In mouse luteinized-granulosa cells (MGLC), ATP induces an increase in intracellular Ca2+ concentration by stimulating phospholipase C (PLC) associated with purinergic receptors, leading to production of inositol 1,4,5-trisphosphate (IP3) and subsequent release of Ca2+ from intracellular stores. In this study, we examined the cross-talk between the ryanodine receptors (RyR) and IP3 receptors (IP3R) in response to ATP in MGLC. Specifically, the effect of RyR modulators on ATP response was examined. The results showed that ATP-induced intracellular calcium elevation was abolished by inhibitors of the RyR, such as dantrolene (25 microM) and ryanodine (80 microM). When the MGLC were stimulated with activators of RyR, 2 microM ryanodine and 10 mM caffeine, the ATP-elicited response was decreased. These actions were independent of IP3 production stimulated by ATP. Hence, ATP-induced intracellular Ca2+ mobilization involves the coordinated action of both types of calcium release channels (CRCs). Using fluorescent probes, it was shown that IP3R is uniformly distributed throughout the cell; in contrast, RyR is mainly found around the nuclei. It is concluded that the IP3R and the RyR are functionally associated, and both play a role in the pattern of Ca2+ increase observed during purinergic stimulation of MGLC. This coupling may provide a highly efficient amplification mechanism for ATP stimulation of Ca2+ mobilization.     

ATP stimulates calcium-dependent glutamate release from cultured astrocytes

ATP caused a dose-dependent, receptor-mediated increase in the release of glutamate and aspartate from cultured astrocytes. Using calcium imaging in combination HPLC we found that the increase in intracellular calcium coincided with an increase in glutamate and aspartate release. Competitive antagonists of P(2) receptors blocked the response to ATP. The increase in intracellular calcium and release of glutamate evoked by ATP were not abolished in low Ca(2+)-EGTA saline, suggesting the involvement of intracellular calcium stores. Pre-treatment of glial cultures with an intracellular Ca(2+) chelator abolished the stimulatory effects of ATP. Thapsigargin (1 microM), an inhibitor of Ca(2+)-ATPase from the Ca(2+) pump of internal stores, significantly reduced the calcium transients and the release of aspartate and glutamate evoked by ATP. U73122 (10 microM, a phospholipase C inhibitor, attenuated the ATP-stimulatory effect on calcium transients and blocked ATP-evoked glutamate release in astrocytes. Replacement of extracellular sodium with choline failed to influence ATP-induced glutamate release. Furthermore, inhibition of the glutamate transporters p-chloromercuri-phenylsulfonic acid and Ltrans-pyrolidine-2,4-dicarboxylate failed to impair the ability of ATP to stimulate glutamate release from astrocytes. However, an anion transport inhibitor, furosemide, and a potent Cl(-) channel blocker, 5-nitro-2(3-phenylpropylamino)-benzoate, reduced ATP-induced glutamate release. These results suggest that ATP stimulates excitatory amino acid release from astrocytes via a calcium-dependent anion-transport sensitive mechanism.     

Sustained calcium entry through P2X nucleotide receptor channels in human airway epithelial cells

Purinergic receptor stimulation has potential therapeutic effects for cystic fibrosis (CF). Thus, we explored roles for P2Y and P2X receptors in stably increasing [Ca(2+)](i) in human CF (IB3-1) and non-CF (16HBE14o(-)) airway epithelial cells. Cytosolic Ca(2+) was measured by fluorospectrometry using the fluorescent dye Fura-2/AM. Expression of P2X receptor (P2XR) subtypes was assessed by immunoblotting and biotinylation. In IB3-1 cells, ATP and other P2Y agonists caused only a transient increase in [Ca(2+)](i) derived from intracellular stores in a Na(+)-rich environment. In contrast, ATP induced an increase in [Ca(2+)](i) that had transient and sustained components in a Na(+)-free medium; the sustained plateau was potentiated by zinc or increasing extracellular pH. Benzoyl-benzoyl-ATP, a P2XR-selective agonist, increased [Ca(2+)](i) only in Na(+)-free medium, suggesting competition between Na(+) and Ca(2+) through P2XRs. Biochemical evidence showed that the P2X(4) receptor is the major subtype shared by these airway epithelial cells. A role for store-operated Ca(2+) channels, voltage-dependent Ca(2+) channels, or Na(+)/Ca(2+) exchanger in the ATP-induced sustained Ca(2+) signal was ruled out. In conclusion, these data show that epithelial P2X(4) receptors serve as ATP-gated calcium entry channels that induce a sustained increase in [Ca(2+)](i). In airway epithelia, a P2XR-mediated Ca(2+) signal may have therapeutic benefit for CF.     

Effect of erythromycin on ATP-induced intracellular calcium response in A549 cells

ATP induced a biphasic increase in the intracellular Ca(2+)concentration ([Ca(2+)](i)), an initial spike, and a subsequent plateau in A549 cells. Erythromycin (EM) suppressed the ATP-induced [Ca(2+)](i) spike but only in the presence of extracellular calcium (Ca(2+)(o)). It was ineffective against ATP- and UTP-induced inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] formation and UTP-induced [Ca(2+)](i) spike, implying that EM perturbs Ca(2+) influx from the extracellular space rather than Ca(2+)release from intracellular Ca(2+) stores via the G protein-phospholipase C-Ins(1,4,5)P(3) pathway. A verapamil-sensitive, KCl-induced increase in [Ca(2+)](i) and the Ca(2+) influx activated by Ca(2+) store depletion were insensitive to EM. 3'-O-(4-benzoylbenzoyl)-ATP evoked an Ca(2+)(o)-dependent [Ca(2+)](i) response even in the presence of verapamil or the absence of extracellular Na(+), and this response was almost completely abolished by EM pretreatment. RT-PCR analyses revealed that P2X(4) as well as P2Y(2), P2Y(4), and P2Y(6) are coexpressed in this cell line. These results suggest that in A549 cells 1) the coexpressed P2X(4) and P2Y(2)/P2Y(4) subtypes contribute to the ATP-induced [Ca(2+)](i) spike and 2) EM selectively inhibits Ca(2+) influx through the P2X channel. This action of EM may underlie its clinical efficacy in the treatment of airway inflammation.     

Ca2+ store depletion and endoplasmic reticulum stress are involved in P2X7 receptor-mediated neurotoxicity in differentiated NG108-15 cells

P2X7 receptor (P2X7R) activation by extracellular ATP triggers influx of Na(+) and Ca(2+), cytosolic Ca(2+) overload and consequently cytotoxicity. Whether disturbances in endoplasmic reticulum (ER) Ca(2+) homeostasis and ER stress are involved in P2X7R-mediated cell death is unknown. In this study, a P2X7R agonist (BzATP) was used to activate P2X7R in differentiated NG108-15 neuronal cells. In a concentration-dependent manner, application of BzATP (10-100 μM) immediately raised cytosolic Ca(2+) concentration ([Ca(2+)]i) and caused cell death after a 24-h incubation. P2X7R activation for 2 h did not cause cell death but resulted in a sustained reduction in ER Ca2+ pool size, as evidenced by a diminished cyclopiazonic acid-induced Ca(2+) discharge (fura 2 assay) and a lower fluorescent signal in cells loaded with Mag-fura 2 (ER-specific Ca(2+)-fluorescent dye). Furthermore, P2X7R activation (2 h) led to the appearance of markers of ER stress [phosphorylated α subunit of eukaryotic initiation factor 2 (p-eIF2α) and C/EBP homologous protein (CHOP)] and apoptosis (cleaved caspase 3). Xestospongin C (XeC), an antagonist of inositol-1,4,5-trisphosphate (IP3) receptor (IP3R), strongly inhibited BzATP-triggered [Ca(2+)]i elevation, suggesting that the latter involved Ca(2+) release via IP3R. XeC pretreatment not only attenuated the reduction in Ca(2+) pool size in BzATP-treated cells, but also rescued cell death and prevented BzATP-induced appearance of ER stress and apoptotic markers. These novel observations suggest that P2X7R activation caused not only Ca(2+) overload, but also Ca(2+) release via IP3R, sustained Ca(2+) store depletion, ER stress and eventually apoptotic cell death.     

Comparison of the effect of ATP on intracellular calcium ion dynamics between rat testicular and cerebral arteriole smooth muscle cells

Adenosine 5'-triphosphate (ATP), when released from neuronal and non-neuronal tissues, interacts with cell surface receptors produces a broad range of physiological responses. The goal of the present study was to examine the issue of whether vascular smooth muscle cells respond to ATP. To this end, the dynamics of the intracellular concentration of calcium ions ([Ca(2+)](i)) in smooth muscle cells in testicular and cerebral arterioles was examined by laser scanning confocal microscopy. ATP produced an increase in [Ca(2+)](i) in arteriole smooth muscle cells. While P1 purinoceptor agonists had no effect on this process, P2 purinoceptor agonists induced a [Ca(2+)](i) increase and a P2 purinoceptor antagonist, suramin, completely inhibited ATP-induced [Ca(2+)](i) dynamics in both arteriole smooth muscle cells.In testicular arterioles, Ca(2+) channel blockers and the removal of extracellular Ca(2+), but not thapsigargin pretreatment, abolished the ATP-induced [Ca(2+)](i) dynamics. In contrast, Ca(2+) channel blockers and the removal of extracellular Ca(2+) did not completely inhibit ATP-induced [Ca(2+)](i) dynamics in cerebral arterioles. Uridine 5'-triphosphate caused an increase in [Ca(2+)](i) only in cerebral arterioles and alpha,beta-methylene ATP caused an increase in [Ca(2+)](i) in both testicular and cerebral arterioles.We conclude that testicular arteriole smooth muscle cells respond to extracellular ATP via P2X purinoceptors and that cerebral arteriole smooth muscle cells respond via P2X and P2Y purinoceptors.     

Ca2+ regulation in detrusor smooth muscle from developing fetal sheep bladders

Sheep fetus is a useful model to study in utero bladder outflow obstruction but little is known about cell physiology of fetal bladders. To remedy this defect we have characterised intracellular Ca(2+) regulation in fetal sheep myocytes of different developmental ages. Fetal detrusor myocytes had a similar resting [Ca(2+)](i) to adult cells and exhibited transient [Ca(2+)](i) increases in response to carbachol, ATP, high-K, caffeine and low-Na. The carbachol transients were abolished by atropine and caffeine; the ATP response was blocked by alpha,beta-methylene ATP; high-K-evoked [Ca(2+)](i) rises were antagonised by verapamil. The maximal responses to carbachol, high-K, caffeine and low-Na in fetal cells were similar to those of adult counterparts, whilst the ATP response was smaller (p < 0.05). These variables were largely similar between the three gestational groups with the exception of ATP-induced response between early fetal and adult bladders (p < 0.05). Dose-response curves to carbachol demonstrated an increase of potency between mid-gestation and early adulthood (p < 0.05). These data show that muscarinic receptors coupled to intracellular Ca(2+) release, P2X receptor-linked Ca(2+) entry, depolarisation-induced Ca(2+) rise via L-type Ca(2+) channels, Na(+)/Ca(2+) exchange and functional intracellular Ca(2+) stores are all operational in fetal bladder myocytes. Whilst most of Ca(2+) regulators are substantially developed and occur at an early fetal age, a further functional maturation for cholinergic sensitivity and purinergic efficacy continues throughout to adulthood.     

Purinergic activation of Ca2+-permeable TRPV4 channels is essential for mechano-sensitivity in the aldosterone-sensitive distal nephron

Mechanical forces are known to induce increases of [Ca(2+)](i) in the aldosterone-sensitive distal nephron (ASDN) cells to regulate epithelial transport. At the same time, mechanical stress stimulates ATP release from ASDN cells. In this study, we combined ratiometric Fura-2 based monitoring of [Ca(2+)](i) in freshly isolated split-opened ASDN with targeted deletion of P2Y2 and TRPV4 in mice to probe a role for purinergic signaling in mediating mechano-sensitive responses in ASDN cells. ATP application causes a reproducible transient Ca(2+) peak followed by a sustained plateau. Individual cells of the cortical collecting duct (CCD) and the connecting tubule (CNT) respond to purinergic stimulation with comparative elevations of [Ca(2+)](i). Furthermore, ATP-induced Ca(2+)-responses are nearly identical in both principal (AQP2-positive) and intercalated (AQP2-negative) cells as was confirmed using immunohistochemistry in split-opened ASDN. UTP application produces elevations of [Ca(2+)](i) similar to that observed with ATP suggesting a dominant role of P2Y2-like receptors in generation of [Ca(2+)](i) response. Indeed, genetic deletion of P2Y2 receptors decreases the magnitude of ATP-induced and UTP-induced Ca(2+) responses by more than 70% and 90%, respectively. Both intracellular and extracellular sources of Ca(2+) appeared to contribute to the generation of ATP-induced Ca(2+) response in ASDN cells. Importantly, flow- and hypotonic-induced Ca(2+) elevations are markedly blunted in P2Y2 -/- mice. We further demonstrated that activation of mechano-sensitive TRPV4 channel plays a major role in the sustained [Ca(2+)](i) elevation during purinergic stimulation. Consistent with this, ATP-induced Ca(2+) plateau are dramatically attenuated in TRV4 -/- mice. Inhibition of TRPC channels with 10 μM BTP2 also decreased ATP-induced Ca(2+) plateau whilst to a lower degree than that observed with TRPV4 inhibition/genetic deletion. We conclude that stimulation of purinergic signaling by mechanical stimuli leads to activation of TRPV4 and, to a lesser extent, TRPCs channels, and this is an important component of mechano-sensitive response of the ASDN.     

ATP increases intracellular calcium in supraoptic neurons by activation of both P2X and P2Y purinergic receptors

ATP increases intracellular calcium concentration ([Ca(2+)](i)) in supraoptic nucleus (SON) neurons in hypothalamo-neurohypophyseal system explants loaded with the Ca(2+)-sensitive dye, fura 2-AM. Involvement of P2X purinergic receptors (P2XR) in this response was anticipated, because ATP stimulation of vasopressin release from hypothalamo-neurohypophyseal system explants required activation of P2XRs, and activation of P2XRs induced an increase in [Ca(2+)](i) in dissociated SON neurons. However, the ATP-induced increase in [Ca(2+)](i) persisted after removal of Ca(2+) from the perifusate ([Ca(2+)](o)). This suggested involvement of P2Y purinergic receptors (P2YR), because P2YRs induce Ca(2+) release from intracellular stores, whereas P2XRs are Ca(2+)-permeable ion channels. Depletion of [Ca(2+)](i) stores with thapsigargin (TG) prevented the ATP-induced increase in [Ca(2+)](i) in zero, but not in 2 mM [Ca(2+)](o), indicating that both Ca(2+) influx and release of intracellular Ca(2+) contribute to the ATP response. Ca(2+) influx was partially blocked by cadmium, indicating a contribution of voltage-gated Ca(2+) channels. PPADS (pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid), and iso-PPADS, P2XR antagonists, attenuated, but did not abolish, the ATP-induced increase in [Ca(2+)](i). Combined treatment with PPADS or iso-PPADS and TG prevented the response. A cocktail of P2YR agonists consisting of UTP, UDP, and 2-methylthio-ADP increased [Ca(2+)](i) (with or without tetrodotoxin) that was markedly attenuated by TG. 2-Methylthio-ADP alone induced consistent and larger increases in [Ca(2+)](i) than UTP or UDP. MRS2179, a specific P2Y(1)R antagonist, eliminated the response to ATP in zero [Ca(2+)](o). Thus, both P2XR and P2YR participate in the ATP-induced increase in [Ca(2+)](i), and the P2Y(1)R subtype is more prominent than P2Y(2)R, P2Y(4)R, or P2Y(6)R in SON.     

A new mode of Ca2+ signaling by G protein-coupled receptors: gating of IP3 receptor Ca2+ release channels by Gbetagamma

The most common form of Ca(2+) signaling by Gq-coupled receptors entails activation of PLCbeta2 by Galphaq to generate IP(3) and evoke Ca(2+) release from the ER. Another form of Ca(2+) signaling by G protein-coupled receptors involves activation of Gi to release Gbetagamma, which activates PLCbeta1. Whether Gbetagamma has additional roles in Ca(2+) signaling is unknown. Introduction of Gbetagamma into cells activated Ca(2+) release from the IP(3) Ca(2+) pool and Ca(2) oscillations. This can be due to activation of PLCbeta1 or direct activation of the IP(3)R by Gbetagamma. We report here that Gbetagamma potently activates the IP(3) receptor. Thus, Gbetagamma-triggered [Ca(2+)](i) oscillations are not affected by inhibition of PLCbeta. Coimmunoprecipitation and competition experiments with Gbetagamma scavengers suggest binding of Gbetagamma to IP(3) receptors. Furthermore, Gbetagamma inhibited IP(3) binding to IP(3) receptors. Notably, Gbetagamma activated single IP(3)R channels in native ER as effectively as IP(3). The physiological significance of this form of signaling is demonstrated by the reciprocal sensitivity of Ca(2+) signals evoked by Gi- and Gq-coupled receptors to Gbetagamma scavenging and PLCbeta inhibition. We propose that gating of IP(3)R by Gbetagamma is a new mode of Ca(2+) signaling with particular significance for Gi-coupled receptors.